ALBERT

Description: Background: Acute myeloid leukemia (AML) is most common in people over the age of 60 where it remains an almost incurable malignancy with a grim prognosis. Evaluation of new therapeutic agents in vitro and in vivo is critical for drug development, yet there are few in-vivo models for studying human leukemia and its therapy. The best model used is the high cost immune-deficient mice and that require several weeks to assess drug response. To complicate matters, AML almost certainly relapses with cells that are not necessarily exactly identical to the original malignant clone, often limiting therapeutic options. The development of anti-leukemia therapies could be facilitated by a rapid and cost effective in vivo system for evaluating response to new drugs. Additionally, decisions regarding personalized treatment for relapsed or refractory leukemia patients must be rapid, and produce results within several days, as longer time periods can be detrimental. Herein, we describe a fast, economical, in ovo turkey embryo model, which provides a unique system to meet these requirements. The model can be used for the assessment of human leukemia infiltration in medullary and extramedullary tissues and more importantly for rapid testing of anti- leukemic agents within the bone marrow (BM). This system can be applied for new drug development and for personalized real time response of patient cells to potential leukemia therapies. Methods: BCR/Abl+ AML lines K562 and LAMA-84 , c-Kit+ CHRF-4288, fresh AML patient and Raji Burkit lymphoma cells (5x106) were injected into turkey egg chorioallantoic membrane (CAM) veins on embryonic day E11 previously optimized (1). Engraftment in BM was detected by flow cytometry (FC) using anti-human CD71 or anti- human CD33 for AML and anti human CD45 for Raji cells, or by Quantitative real time PCR (Q-PCR) comparing the amount of genomic human to the amount of avian DNA and number of human /avian cells in BM. Drug response was tested by IV injection of therapeutic range doses of Imatinib (Glivec ®), Doxorubicin or dexamethasone, 48H after grafting cells. Drug levels were precalibrated to be non-toxic to the developing embryo by LD50 and BM cell viability compared to control (Taizi M et al Exp Hem 34:1698,2006, Grinberg I et al, Leuk Res. 33:1417, 2009). Six days later (E19) the embryos were sacrificed and the BM collected for FC and hematopoietic and non-hematopoietic tissues for molecular analysis. Results: The kinetics of leukemia cell engraftment in the BM on E15, E18, and E23 in BM and liver after cell injection on day E11 was assessed to determine the optimal treatment and readout times. The highest engraftment level in BM and liver was detected at E18 by Q-PCR, and FC in more than 90% of the injected embryos. We quantitatively compared the engraftment of AML cells at E18-20 without and with drug treatment that was administered IV 48 hours after cell injection. The average engraftment (±SD.) in the BM after one week was 4.5%+1.7 K562, 5.83% +0.88 LAMA-84, 11.2%+3.5 CHRF-4288, 8.9% +1.6 Raji (n=7-15 per group) and 2.5% fresh leukemia cells detected by FC and confirmed by Q-PCR. A single dose of either 0.75 mg Imatinib or 50 mg Doxorubicin /embryo previously calibrated to be non toxic to the embryos reduced engraftment of AML cells in BM and in several other organs by more than ten fold. A similar effect was also obtained by a single dose of 5mg dexamethasone in Raji injected embryos. Treatment with 0.5 mg Imatinib on injected ARH-77 (multiple myeloma) or Raji cells had no effect on cell engraftment, while treatment with a single non toxic dose of Revlimid as previously described eliminated engraftment of ARH77 cells (1), clearly demonstrating the specificity of the drugs and utility of the system. Conclusion: Our study demonstrates the potential utility of a practical and unique avian embryo model for testing drug activity on human AML cells in vivo. This system, under preclinical development, is expected to provide a new xenograft platform for real time affordable testing leukemia therapies. More importantly, this may open a new venue for individualized screening for response or resistance to specific therapeutic agents for the relapsed or refractive patient and may lead to better optimization of practical and applicable therapeutic strategies. Disclosures No relevant conflicts of interest to declare.

Description: A novel therapeutic approach in cancer attempting to stimulate host anti-tumor immunity involves blocking of immune checkpoints. Lymphocyte-activation gene 3 (LAG3) is an immune checkpoint receptor expressed on activated/exhausted T-cells. When engaged by MHC Class II (MHCII) molecules, LAG3 negatively regulates T-cell function thereby contributing to tumor escape. Intriguingly, a soluble LAG3 variant was reported to activate both immune and malignant MHCII-presenting cells. In this study, we examined the role of LAG3 in the pathogenesis of CLL and show that CLL cells express and secrete LAG3. High levels of surface and soluble LAG3 were associated with the unmutated immunoglobulin variable heavy chain CLL and a more aggressive disease. It was evident that recombinant soluble LAG3-Ig fusion protein activated CLL cells, induced increased levels of Bcl-2 and Mcl-1 and protected the cells from spontaneous apoptosis, utilizing a mechanism mediated through MHCII engagement. Our data suggest that autocrine secretion of soluble LAG3 from CLL cells and its subsequent interaction with MHCII on their surface, promotes CLL cell activation and anti-apoptotic effects. Moreover, CLL cells may impose immune exhaustion on their microenvironment by expressing MHCII, thus affecting the surrounding, LAG3-presenting immune cells. Hence, blocking LAG3-MHCII interactions is a potential therapeutic target in CLL. Disclosures Avivi: Tel Aviv Sourasky Medical center: Consultancy, Other: consultancy to :BMS Roche. Wiestner:Pharmacyclics: Research Funding; Acerta Pharma: Research Funding.

Description: In the last decade, the B-cell receptor (BCR) has emerged as a pivotal stimulus in CLL pathogenesis. The BCR responsiveness in CLL cells is heterogeneous among patients and correlates with disease aggressiveness. Here we show for the first time, that SLP76 a key scaffold protein in T-cell receptor (TCR) signaling, is ectopically expressed in CD19+ purified CLL cells (purity 〉95%) in the majority of patients, and is co-expressed with other components of the TCR pathway, including LCK and ZAP70. SLP76 mRNA levels correlated with its protein expression and SLP76 protein levels were higher in unmutated IGHV and ZAP70+ CLL cells. SLP76 was found to be functionally active in CLL cells, as it becomes phosphorylated in response to BCR engagement in a time dependent manner. Activation with anti-IgM antibody results in phosphorylation of SLP76 on the positive regulatory tyrosine residue Y128 residue with increased physical association with Btk, peaking after 15 minutes. The negative regulatory residue S376 became phosphorylated only after 45', concomitantly with downregulation of the tyrosine phosphorylation. SLP76 phosphorylation in response to BCR engagement did not correlate with total ZAP70 expression. Pre-incubation of CLL cells with the LCK kinase inhibitor LCKi and with the SYK inhibitor R406, inhibited SLP76 phosphorylation in response to BCR activation, while the BTK inhibitor-ibrutinib had no effect. These suggest that LCK and SYK, but not ZAP70, play a central role in the upstream signaling involved in SLP76 activation in CLL cells. Knockdown of SLP76 in CLL cells resulted in decreased induction of BTK and PLCγ2 phosphorylation after BCR activation with anti-IgM. Consistent with our findings that SLP76 in involved in BCR signaling in CLL cells, we found that high total SLP76 expression was associated with a shorter time to first progression or need for treatment. In conclusion, SLP76 is ectopically expressed in CLL cells and plays a role in BCR signaling in those cells. Disclosures No relevant conflicts of interest to declare.

Description: Uteroplacental thrombosis has been implicated in the pathogenesis of fetal loss, intrauterine growth restriction (IUGR) and preeclampsia. The role of inherited and acquired thrombophilias in such pregnancy complications is controversial. Recently, increased levels of circulating procoagulant microparticles (MPs) were related to fetal loss and preeclampsia (Laude et al. Thromb Haemost2001;85:18, Gonzalez-Quintero et al. Am J Obstet Gynecol2004;191:1418)). In this study, we measured the level of MPs derived from endothelial cells, platelets, and monocytes by flow cytometry using monoclonal antibodies against CD31, CD41 and CD14, respectively in 262 healthy unrelated primipara with a spontaneous singleton pregnancy. Expression of tissue factor by a monoclonal antibody and phosphatidyl serine by annexin 5 on MPs were also measured. All women were followed from the 24th week of gestation until delivery and IUGR, small for gestational age (SGA) at birth, pregnancy induced hypertension (PIH) or preeclampsia (PE) or none of the above were recorded. Blood sampling was carried out at 24th week of gestation. The levels of CD31 and CD41- positive MPs in women with and without the above pregnancy complications are presented in the table. Not shown are values of CD14, annexin 5 or tissue factor on MPs since their median levels were negligible in all study groups. There was no statistically significant difference in CD31 and CD41- positive MPs between women who developed or not the indicated pregnancy complications. Also, there was no significant difference in levels of these MPs between women with and without the common inherited thrombophilias, factor V Leiden and or prothrombin mutation (28 patients) and lupus anticoagulant (10 patients). These data indicate that measurement of the level of MP at the 24th week of gestation has no predictive value for development of PIH, PE, IUGR or SGA. CD31 CD41 Number Median % p Median % p PIH or PE No 226 1.50 0.22 1.80 PIH Yes 13 1.57 3.07 0.53 PE Yes 22 0.62 1.87 IUGR No 207 1.53 0.13 1.95 0.08 IUGR Yes 32 1.30 1.71 SGA No 232 1.48 0.64 1.85 0.81 SGA Yes 26 1.27 2.24

Description: Multiple myeloma (MM) is an incurable malignancy of terminally differentiated B cells with heterogeonous clinical presentation. Although MM is viewed clinically as a disease restricted to the bone marrow, it is considered to be a systemic malignancy and peripheral blood MM cells can be detected in the majority of patients at diagnosis. In an attempt to clarify the diverse clinical presentation of this disease, we hypothesized that MM cells comprise a heterogeneous cell population which can be distinguished by their motile and adhesive phenotype: while most of the cells strongly adhere to the bone marrow environment, other non-adhesive sub-populations are able to invade into adjacent sites within the bone marrow, or circulate to distant locations. In order to study the motile and adhesive diversity within malignant plasma cell populations we used the ARH77 plasma cell leukemia cell line. These cells were fractionated into adherent and poorly-adherent sub-populations by continuous passages on fibronectin coated tissue culture plates. The enriched sub-populations were the basis for further experiments of adhesion, motility, cytoskeletal organization and flow cytometric profile. The adhesive sub-population readily attached and spread on fibronectin, forming paxillin-rich focal adhesions. In contrast, the poorly-adherent sub-population attached loosely to fibronectin, did not spread and failed to organize paxillin into discrete structures. The differences between these sub-populations of ARH77 cells were not observed when the cells were plated on hyaluronan or control, uncoated dish, indicating that the differences are in the integrin-mediated adhesion system. Although the flow cytomentry analysis indicated that the surface expression of integrins b1, a5 and aV is identical in the two sub-populations, specific inhibitory anti-integrin a5b1 antibodies inhibited the poorly-adherent sub-population, without significantly affecting the highly adhesive sub-population. These results suggest different mechanisms that regulate the adhesive interactions of the two sub-populations. To elucidate the motile phenotype of these cells, the two sub-populations were plated on fibronectin and hyaluronan. The migration velocity of the poorly-adherent cells on fibronectin was significantly higher compared to that of the adhesive cells. Moreover, the adhesive sub-population exhibited higher binding to physiologically-relevant substrates (cultured stromal cells and bone fragments). Furthermore, flow cytometry indicated that while both sub-populations expressed similar levels of the Ig kappa light chain, the adhesive sub-population expressed significantly higher levels of CD44 and CD45 and lower levels of CD138, compared to the poorly-adhesive population. In conclusion, we have shown that MM cells differ by their adhesive and motile properties and can be identified and separated according to their flow cytometric profile, thus providing useful tools for studying the differential properties of the various MM sub-populations in patients, and their clinical relevance. Moreover, this molecular diversity of MM cells can be further employed to design new drugs that can specifically inhibit the survival and dissemination of clinically important sub-populations.

Description: Abstract 3577 Emerging data on intra-clonal diversity imply that this phenomenon may play a role in the clinical outcome of patients with CLL. Subsets of the CLL clone that respond more robustly to external stimuli may gain a growth and survival advantage which may possibly promote clonal evolution. Here we report an intra-clonal diversity in response to CD19 engagement in CLL cells identified by using a non-conventional flow cytometric analysis. Monoclonal anti-CD19 antibody rapidly induced cellular aggregation of CLL cells identified as a high side scatters lymphoid population (HSLP). The percentage of the HSLP within the whole CLL population was highly reproducible in individual patients, but was extremely variable between patients (range 0–39.4%, median-19.95%). This inter-individual heterogeneity was attributed to the intrinsic ratio between CD19 responsive and unresponsive CLL subsets and was seen to correlate with time to treatment (TTT), but not with ZAP-70 expression. During a median follow-up period of 45.6 months (n=46), CLL patients with more than 20% of HSLP had a median TTT of 50.7 months from the time of diagnosis while this median has yet not been reached in patients with a lower proportion of HSLP (p=0.019). The rapid induction of cellular aggregation via CD19 was completely abrogated by disruption of the cholesterol-rich plasma membrane rafts by methyl-cyclodextrin, but minimally affected by protein kinase and actin cytoskeleton inhibitors. The relatively ineffective blocking of cellular aggregation by cytoplasmatic signaling inhibitors suggests that this response is mainly attributed to CD19 association with other trans-membrane components, which possibly occurs within the lipid rafts. The CLL sub-population which responded to CD19 clustering expressed higher mRNA levels of c-Myc, and exhibited different morphological features from the non-responsive CLL cells subset. The CD19-responsive subset also correlated with higher CD20 expression and a marked decrease in HSLP was noted in samples taken 24 h after rituximab administration (n=6). As expected, rituximab resulted in a significant decrease in the total CLL counts, with the specific elimination of the HSLP. Comparison of HSLP induction by CD19 in normal B-cells and in leukemic cells from B-type ALL revealed very low levels of HSLP in B-cells from healthy individuals and B-ALL (n=5, range 0.5–2.3%, median-0.9%). Leukemic cells derived from other indolent B-cell lymphoproliferative disorders (non-Hodgkin lymphomas and hairy cell leukemia) had the highest proportion of HSLP of all samples screened (n=8, range 34–60%, median-38.3%). Conclusions: we have identified intra-clonal diversity for CD19-mediated cellular aggregation in CLL which correlates with time to treatment initiation. This phenomenon was completely lipid raft dependent and restricted to a distinct CLL cell subset. The heterogeneous CD19 response may reflect basic differences in the juxta-membrane signaling patterns within the CLL clone. Disclosures: No relevant conflicts of interest to declare.

Description: While umbilical cord blood provides an important source of hematopoietic stem/progenitor cells (HSPC) for allogeneic transplantation in children, its use in adults is limited by the inadequate number of primitive stem cells and megakaryocyte progenitors (Mk-p) in single or even double CB units resulting in prolonged thrombocytopenia. Thrombopoietin treatment is not effective in these patients due to the paucity of target progenitors and patients require multiple platelet transfusions until the long-term engrafting cells can support thrombopoiesis, thus new modalities to increase progenitor cell dose are needed. A new transplantation strategy could involve the infusion of ex vivo-generated Mk-p together with unmanipulated single or double CB units. While CB CD34+ cells can be expanded by several reported methods, these rare cells cannot be sacrificed from the CB units due to their limited number. We propose a novel ex-vivo strategy to facilitate HSPC and Mk-p expansion from mononuclear cells (MNC) of a small aliquot of CB using conditions that mimic the hematopoietic niche, in short term cultures. Fibronectin (FN) was considered to be a prime candidate to support proliferation because it is a major extracellular matrix (ECM) component of all bone marrow hematopoietic microenvironments which is known to enhance viability and proliferation of HSPC. Other growth stimulators added were thrombopoietin (r-hu-TPO), the major physiological stimulator of MK and the synthetic hematopoietic stress peptide ARP derived from acetylcholinesterase, shown to increase transplantable Mk-p and produce human platelets in NOD/SCID mice (Pick et al, Blood 2006, Grisaru et al, J Imm 2006). High definition flow cytometry enabled assessing expansion of the SSClow/CD34high HSPC, and the SSClow/CD45dim/neg/CD41high Mk-p, and their subpopulations on day 0 and 10 of culture. True MK expansion was assessed by gating out of granulocyte and monocytes, which acquire CD41+ adherent platelets in culture. FN alone increased viability and expansion of HSPC by 6.9 fold and MK-p by 4-fold, while r-hu-TPO alone enhanced Mk-p proliferation with an average expansion of 8.3-fold in agreement with its known activity. Combining FN with r-hu-TPO produced a 25-fold increase in the number of MK-p while adding ARP to FN and r-hu-TPO was even more powerful, doubling the number of cells with a highly significant average expansion of 59-fold (p 〈 0.001). To define the progenitor subpopulations that contributed to Mk-p proliferation with FN, r-hu-TPO and ARP, we further analyzed the resulting subsets of MK-p cells, which also expressed either CD34, or the early myeloid marker CD33. The CD41high/CD34high population was increased by 4 fold, while the CD41high/CD33+ Mk-p, a subset with properties similar to clonogenic GEMM progenitors that could provide both myeloid and megakaryocytic cells post-transplant, were stimulated 30–50 fold. This notion is confirmed by the stimulation of CFU-MK and CFU-GEMM obtained under these conditions. Considering that expansion of MK-p requires proliferation of the HSPC precursor, we examined the proliferation of CD34+ progenitor cells and their subpopulations; CD34high/CD33+ or CD34high/CD41low uncommitted HSPC and CD41 high committed Mk subpopulations. The addition of FN alone stimulated CD34+ HSPC expansion by 6.9-fold (p 〈 0.05). All cultures that contained the ARP peptide maintained a high proliferation capacity, confirming that ARP protects and drives CD34+HSPC and early myeloid cell proliferation (Deutsch et al Exp Hem 2002). The addition of r-hu-TPO and ARP to FN produced a synergistic proliferative effect on the CD34+/CD41low HSPC stimulating a dramatic 440 fold increase of these uncommitted cells. These data support the notion that FN is protective and plays an essential role in enabling HSPC and MK-p expansion driven by r-hu-TPO and ARP. These conditions also supported MK maturation, as measured by increased high ploidy cells and elevated expression of GPIIb/IIIa detected by quantitative real time PCR. We demonstrate that expansion of both very early myeloid and Mk-p from a small fraction of the CB unit in short term cultures under conditions that mimic the hematopoietic niche is feasible, easy to perform and can comply with GTP requirements. This approach may lead to the development of more effective cell therapy modalities to facilitate myelopoiesis and platelet production following CBT.