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Novel targeted therapies improve the survival of specific subgroups (defined by genetic variants) of patients with acute myeloid leukemia (AML), validating the paradigm of molecularly targeted therapy. However, identifying correlations between AML molecular attributes and effective therapies is challenging. Recent advances in highthroughput, in vitro drug sensitivity screening applied to primary AML blasts were used to uncover such correlations; however, these methods cannot predict the response of leukemic stem cells. Our study aimed to predict in vitro response to targeted therapies, based on molecular markers, with subsequent validation in leukemic stem cells. We performed ex vivo screening of sensitivity to 46 drugs on 29 primary AML samples at diagnosis or relapse. Using unsupervised hierarchical clustering analysis we identified a group with sensitivity to several tyrosine kinase inhibitors, including the multi-tyrosine kinase inhibitor, dasatinib, and searched for correlations between the response to dasatinib, exome sequencing and gene expression in our dataset and in the Beat AML dataset. Unsupervised hierarchical clustering analysis of gene expression resulted in clustering of dasatinib responders and non-responders. In vitro response to dasatinib could be predicted based on gene expression (area under the curve=0.78). Furthermore, mutations in FLT3/ITD and PTPN11 were enriched in the dasatinib-sensitive samples as opposed to mutations in TP53 which were enriched in resistant samples. Based on these results, we selected FLT3/ITD AML samples and injected them into NSG-SGM3 mice. Our results demonstrate that in a subgroup of FLT3/ITD AML (4 out of 9) dasatinib significantly inhibited leukemic stem cell engraftment. In summary we show that dasatinib has an anti-leukemic effect both on bulk blasts and, more importantly, on leukemic stem cells from a subset of AML patients that can be identified based on mutational and expression profiles. Our data provide a rational basis for clinical trials of dasatinib in a molecularly selected subset of AML patients.

Background: Venetoclax-based combinations were recently approved to treat patients (pts) with acute myeloid leukemia (AML) ineligible for intensive chemotherapy. Limited prospective 'real-world' data is available on treatment patterns of venetoclax-based therapy in routine clinical practice. We investigated patterns of patient selection, efficacy, toxicity, patient related outcome and post-remission management in a nationwide multicenter prospective observational trial. Methods: Newly diagnosed pts with AML were enrolled at the time of venetoclax-based therapy initiation from 10 medical centers in Israel. Demographic, clinical and patient-related baseline characteristics were documented. Treatment patterns, safety and efficacy outcomes are reported. Results: Between August 12, 2019, and June 17, 2021(data cut) ,127 AML pts were enrolled to receive venetoclax based therapy. Baseline patient and disease characteristics are reported in Table 1. The main reasons for physician's choice of venetoclax-based therapy were age ≥75, comorbidities and ECOG ≥2 (patient related factors) in 76% of cases and adverse disease biology predicting poor response to intensive chemotherapy (disease related factors) in 24% of cases. Most pts started therapy in an inpatient setting, 82 (64.6%) with a median hospitalization duration of 14 days, while 44 pts (34.6%) started therapy as out pts. Pts received a median of 3.8 cycles of therapy (range 1-21). Most pts (97%) received venetoclax in combination with hypomethylating agents. The full dose of 400mg QD after a median ramp-up duration of 3 days was achieved in 88% of the pts. Dose interruptions and dose modifications during follow-up occurred in 59 (46%) and 30 (24%) of pts, respectively. To allow for adequate follow up for response assessment, efficacy analysis was limited to pts enrolled prior to December 31, 2020, and included 108 pts with a median follow-up of 8 months (range 1-20). As of data cut, 93 pts completed cycle 1 of therapy, 66 pts completed cycle 3 and 39 pts completed cycle 6. 29 pts (27%) are still active on treatment. Best composite complete remission [CCR = complete remission (CR) plus CR with incomplete count recovery (CRi)] was achieved in 62 (57%) pts. CCR rates were assessed in different pre-defined subgroups. Best CCR in pts selected for therapy based on disease-related and patient-related factors were 70% and 54% respectively. Best CCR in pts with AML arising from MPN and pts with other AML were 45% and 58% respectively. Estimated median overall survival (OS) of all pts was 9.6 months (range 7.4-10.6) (Figure 1). Achieving CCR was associated with a superior probability for survival. Estimated median OS was 13.6 months (range 10.6 - not reached) in pts achieving CCR and 4.2 months (range 1.2-10.3) in non-CCR (p Allogeneic transplantation following venetoclax based treatment was offered to 16 (26%) pts with a median age of 71 years (range 43-77). Last documented response prior to transplant was CR in 5 (32%) pts, CRi 9 (56%), MLFS 1 (6%) and PR in 1 (6%) patient. Among grade ≥3 AEs were febrile neutropenia in 28% and infections in 21% of pts. Clinical and laboratory tumor lysis syndrome (TLS) was documented in 2 and 4 pts, respectively. Antifungal prophylaxis was administered in 20% of pts and granulocyte colony-stimulating factor (GCSF) support was used in 17% of pts in response. Early death rate at 30 and 60 days were 7% and 13%, respectively. Conclusion: This prospective real-world analysis reveals unique patterns of patient selection and venetoclax treatment utilization in a medical system with wide access for this indication. Venetoclax-based therapies are effective and associated with manageable toxicity, including in AML patient populations that were excluded from previous registration trials with comparable CCR and early death rates. Factors associated with patient selection in the 'real-world' setting and immature follow up data most probably led to a shorter estimated median OS in this analysis as compared to controlled trials. The REVIVE study continues to expand and is expected to provide additional insights on treatment patterns, management as well as clinical and patient related outcomes. Figure 1 Figure 1. Disclosures Wolach: Janssen: Consultancy; Novartis: Consultancy; Amgen: Research Funding; Astellas: Consultancy; Abbvie: Consultancy, Honoraria, Research Funding; Neopharm: Consultancy. Levi: AbbVie: Consultancy, Research Funding. Lavie: AbbVie: Membership on an entity's Board of Directors or advisory committees, Other: Fees for lectures; BMS: Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Other: Fees for lectures; Roche: Other: Fees for lectures; Novartis: Other: Fees for lectures. Tavor: AbbVie: Consultancy. Hellmann: AbbVie: Consultancy. Tadmor: Janssen: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding. Zuckerman: Gilead Sciences: Honoraria, Speakers Bureau; Novartis: Honoraria; Janssen: Honoraria; Cellect Biotechnology: Honoraria; BioSight Ltd: Honoraria; AbbVie: Honoraria; Orgenesis Inc.: Honoraria. Stemer: AbbVie: Consultancy. Berelovich: AbbVie: Current Employment, Current equity holder in publicly-traded company. Ofek: AbbVie: Current Employment, Current equity holder in publicly-traded company. Frankel: AbbVie: Current Employment, Current equity holder in publicly-traded company. Grunspan: AbbVie: Current Employment, Other: May hold equity. Ofran: Medison Israel: Consultancy; Pfizer: Consultancy; Astellas: Consultancy; AbbVie: Consultancy; Janssen: Consultancy. Moshe: Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Lectures; Astellas: Membership on an entity's Board of Directors or advisory committees, Other: Lectures; AbbVie: Membership on an entity's Board of Directors or advisory committees, Other: Lectures.

CXCR4 is a key player in the retention and survival of human acute myeloid leukemia (AML) blasts in the bone marrow (BM) microenvironment. We studied the effects of the CXCR4 antagonist BL-8040 on the survival of AML blasts, and investigated the molecular mechanisms by which CXCR4 signaling inhibition leads to leukemic cell death. Treatment with BL-8040 induced the robust mobilization of AML blasts from the BM. In addition, AML cells exposed to BL-8040 underwent differentiation. Furthermore, BL-8040 induced the apoptosis of AML cells in vitro and in vivo. This apoptosis was mediated by the upregulation of miR-15a/miR-16-1, resulting in downregulation of the target genes BCL-2, MCL-1 and cyclin-D1. Overexpression of miR-15a/miR-16-1 directly induced leukemic cell death. BL-8040-induced apoptosis was also mediated by the inhibition of survival signals via the AKT/ERK pathways. Importantly, treatment with a BCL-2 inhibitor induced apoptosis and act together with BL-8040 to enhance cell death. BL-8040 also synergized with FLT3 inhibitors to induce AML cell death. Importantly, this combined treatment prolonged the survival of tumor-bearing mice and reduced minimal residual disease in vivo. Our results provide a rationale to test combination therapies employing BL-8040 and BCL-2 or FLT3 inhibitors to achieve increased efficacy of these agents.

Background: The outcome of elderly patients with Acute Myeloid Leukemia (AML) is poor and treatment options in these high-risk groups are limited. Recently, venetoclax combinations with hypomethylating agents or low dose cytarabine were approved to treat patients with AML ineligible for intensive chemotherapy. However limited prospective data is available on the safety and efficacy of venetoclax treatment in routine clinical practice. Israel is among the first countries to have approved venetoclax-based combinations as first line therapy for AML and this treatment is fully reimbursed via the national health system. Here we present the initial results of a prospective, multicenter, nationwide trial that sought to assess the use of venetoclax-based therapy in a real-world setting. Methods: A prospective observational nationwide multicenter trial. Newly diagnosed patients with AML were enrolled at the time of venetoclax-based therapy initiation. Demographic, clinical and patient-related baseline characteristics were documented. Treatment patterns, safety and efficacy outcomes are reported. Patient related outcomes were assessed at baseline and after cycle 3 using the EQ-5D-5L and EORTC QLQ-C30 questionnaires. Results: A total of 70 patients were enrolled between August 2019 and June 2020 (data cut off) with a median age of 75 years (range 45-88) and a median follow-up of 74 days (8-232). Two-thirds of patients were males (62.9%). Over one-quarter (28.6%) of patients had an ECOG performance status of 2 or higher; the median modified Charlson Comorbidity Index (CCI) was 0 (range 1-4) with 27.1% with a CCI ≥2. De-novo AML was documented in 44.3%, secondary AML was diagnosed in 52.8% (secondary to MDS (27.1%), MPNs (11.4%) and therapy related AML (14.3%)). European LeukemiaNet (ELN) risk category was favorable, intermediate and adverse in 8.6%, 30% and 42.9%, respectively (Table 1). Time from diagnosis to initiation of therapy was 8 days (median, range 1-38). The main reasons for choosing venetoclax-based low intensity therapy as reported by treating physicians were patient related factors (mainly age>75 years, performance status) in the majority of cases and adverse disease biology predicting poor response to intensive chemotherapy in 17.1%. Of the 57 patients with available data, 38 (67%) initiated therapy in an inpatient setting with a median hospitalization duration of 12 days (range 1-62 days) and 19 (33%) patients started therapy as outpatients. By data cutoff, of 63 patients that initiated therapy 45, 23 and 7 patients completed cycle 1, cycle 3 and cycle 6 assessments, respectively. Complete remission (CR) or CR with incomplete count recovery (CRi) was achieved in 23/44 (52.3%) patients that were assessed for best response. Of responding patients, 6 (23%, 5 CRi and 1 Partial Remission (PR)) went on to receive an allogeneic transplantation (median age 70.5 years). Ninety percent of patients received venetoclax in combination with hypomethylating agents (azacytidine n=56, decitabine n=1). The full dose of 400mg was administered in 87% of cases with a median ramp-up duration of 3 days. Dose interruptions, dose modifications and dose discontinuations during follow-up were frequent and occurred in 41%, 35% and 27%, respectively. During therapy 63.5% of patients experienced adverse events (AE) of any grade; severe AE's were recorded in 41.3% of patients. Febrile neutropenia was documented in 22.2% and Tumor Lysis Syndrome (TLS) was documented in 2 patients (grade 2; 3.2%). Early death rates at 30 and 60 days were 6.3% and 11.1%, respectively. Conclusion: In the real-world setting venetoclax-based therapies are effective and associated with manageable toxicity including in the outpatient setting. In routine practice patient-related factors and disease-related factors (disease-risk) both seem to play a role in choice of therapy. Venetoclax treatment in real-life practice in Israel appears to follow general recommendations, is tolerable with approximately 90% of patients achieving target dose. These observational data are expected to provide information on patient selection patterns, efficacy and safety and patient related outcomes in patients not in clinical trial. Table Disclosures Wolach: AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Fees for lectures and Consultancy, Research Funding; Astellas: Consultancy, Honoraria, Other: Fees for lectures and Consultancy; Pfizer: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Other: Fees for lectures and Consultancy; Amgen: Other: Fees for lectures and Consultancy; Janssen: Other: Fees for lectures and Consultancy. Levi:Abbvie Inc: Consultancy, Research Funding. Canaani:Abbvie: Consultancy, Honoraria, Research Funding. Tadmor:AbbVie: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Sanofi: Consultancy, Speakers Bureau; Medison: Consultancy, Speakers Bureau; Neopharm: Consultancy, Speakers Bureau; 6. Novartis Israel Ltd., a company wholly owned by Novartis Pharma AG: Consultancy, Speakers Bureau. Tavor:Abbvie: Consultancy, Honoraria, Research Funding. Hellmann:Abbvie: Research Funding. Stemer:Abbvie: Research Funding. Cohen:Abbvie Inc: Current Employment, Current equity holder in publicly-traded company. Afik:Abbvie Inc: Current equity holder in publicly-traded company. Ofek:Abbvie Inc: Current Employment. Banayan:Abbvie Inc: Current Employment, Current equity holder in publicly-traded company. Kan:Abbvie Inc: Current Employment, Current equity holder in publicly-traded company. Grunspan:Abbvie Inc: Current Employment, Current equity holder in publicly-traded company. Ofran:AbbVie: Membership on an entity's Board of Directors or advisory committees. Moshe:Astellas: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria, Research Funding.

High-dose cytarabine is the backbone of acute myeloid leukemia (AML) treatment. Nevertheless, its use in older patients is considerably limited due to increased toxicity. BST-236 (INN aspacytarabine) is a novel cytarabine prodrug designed to deliver high-dose cytarabine to target cells with reduced systemic exposure to free cytarabine. This phase 1/2a dose-escalation study was designed to evaluate BST-236 safety, pharmacokinetics, and efficacy in older or unfit-for-intensive-therapy patients with acute leukemia. Twenty-six patients, unfit for standard therapy, who were either relapsed/refractory or newly diagnosed, received BST-236 in 6 dose-escalating cohorts (range 0.3 to 6 g/m(2) per day). BST-236 was administered intravenously once daily over 60 minutes for 6 consecutive days. The median age was 76.5 (26 to 90), with 84.6% of patients ≥70 years. BST-236 was safe and well tolerated. The maximal tolerated dose was 6 g/m(2) per day. Overall response rate was 29.6%. A subgroup analysis of newly diagnosed patients with AML, de novo or secondary to myelodysplastic syndrome, unfit for standard induction (median age 78), demonstrated overall response of 45.5%. The median overall survival was 6.5 months and was not reached in patients achieving complete remission. The findings of this phase 1/2 study suggest that BST-236 safely delivers high and efficacious cytarabine doses to older patients who are unfit for standard induction and lays the foundation for further studies of BST-236 in AML. This trial was registered at www.clinicaltrials.gov as #NCT02544438.

Prevention of central nervous system (CNS) relapse is critical for cure of childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Despite this, mechanisms of CNS infiltration are poorly understood, and the timing, frequency, and properties of BCP-ALL blasts entering the CNS compartment are unknown. We investigated the CNS-engrafting potential of BCP-ALL cells xenotransplanted into immunodeficient NOD.Cg- ITALIC! Prkdc (ITALIC! scid) ITALIC! Il2rg (ITALIC! tm1Wjl)/SzJ mice. CNS engraftment was seen in 23 of 29 diagnostic samples (79%): 2 of 2 from patients with overt CNS disease and 21 of 27 from patients thought to be CNS negative by diagnostic lumbar puncture. Histologic findings mimic human pathology and demonstrate that leukemic cells transit the blood-cerebrospinal fluid barrier situated close to the dural sinuses, the site of recently discovered CNS lymphatics. Retrieval of blasts from the CNS showed no evidence for chemokine receptor-mediated selective trafficking. The high frequency of infiltration and lack of selective trafficking led us to postulate that CNS tropism is a generic property of leukemic cells. To test this, we performed serial dilution experiments which showed CNS engraftment in 5 of 6 mice after transplant of as few as 10 leukemic cells. Clonal tracking techniques confirmed the polyclonal nature of CNS-infiltrating cells, with multiple clones engrafting in both the CNS and periphery. Overall, these findings suggest that subclinical seeding of the CNS is likely to be present in most BCP-ALL patients at original diagnosis, and efforts to prevent CNS relapse should concentrate on effective eradication of disease from this site rather than targeting entry mechanisms.

Ruxolitinib has been shown in two randomized clinical trials to be effective in alleviating systemic symptoms and reducing spleen size in patients with myelofibrosis (MF). We retrospectively evaluated efficacy and tolerability of ruxolitinib in a cohort of unselected MF patients treated in routine clinical practice. One hundred and two patients who began ruxolitinib therapy were identified in 13 participating centers. Ninety three of the patients receiving ruxolitinib for at least 3 months were evaluated for treatment efficacy and toxicity. Median age at ruxolitinib initiation was 67 years. Indications for treatment were constitutional symptoms (15%), symptomatic splenomegaly (6%) or both (76%). Two patients received ruxolitinib for other indications. The median initial ruxolitinib dose was 30 mg/day. Median duration of therapy was 11 months. Eighty two patients (88.2%) responded to therapy, 76 (84.4%) patients had improvement in constitutional symptoms and 60 patients (70.6%) had reduction in spleen length. While on ruxolitinib, 30% of patients had grade 3–4 anemia and 12.9% of patients had grade 3–4 thrombocytopenia. Thirteen patients (14%) discontinued therapy. This analysis of a cohort of MF patients treated with ruxolitinib in routine clinical practice demonstrates the efficacy and tolerability of this drug outside of a highly monitored clinical trial setting.

In acute myeloid leukemia (AML), therapy resistance frequently occurs, leading to high mortality among patients. However, the mechanisms that render leukemic cells drug resistant remain largely undefined. Here, we identified loss of the histone methyltransferase EZH2 and subsequent reduction of histone H3K27 trimethylation as a novel pathway of acquired resistance to tyrosine kinase inhibitors (TKIs) and cytotoxic drugs in AML. Low EZH2 protein levels correlated with poor prognosis in AML patients. Suppression of EZH2 protein expression induced chemoresistance of AML cell lines and primary cells in vitro and in vivo. Low EZH2 levels resulted in derepression of HOX genes, and knockdown of HOXB7 and HOXA9 in the resistant cells was sufficient to improve sensitivity to TKIs and cytotoxic drugs. The endogenous loss of EZH2 expression in resistant cells and primary blasts from a subset of relapsed AML patients resulted from enhanced CDK1-dependent phosphorylation of EZH2 at Thr487. This interaction was stabilized by heat shock protein 90 (HSP90) and followed by proteasomal degradation of EZH2 in drug-resistant cells. Accordingly, inhibitors of HSP90, CDK1 and the proteasome prevented EZH2 degradation, decreased HOX gene expression and restored drug sensitivity. Finally, patients with reduced EZH2 levels at progression to standard therapy responded to the combination of bortezomib and cytarabine, concomitant with the re-establishment of EZH2 expression and blast clearance. These data suggest restoration of EZH2 protein as a viable approach to overcome treatment resistance in this AML patient population.

The Chemokine receptor CXCR4 and its ligand stromal derived factor-1 (SDF-1/CXCL12) are important players involved in cross-talk between leukemia cells and the bone marrow (BM) microenvironment. CXCR4 expression is associated with poor prognosis in AML patients with and without the mutated FLT3 gene. CXCL12 which is constrictively secreted from the BM stroma and AML cells is critical for the survival and retention of AML cells within the BM. In vitro, CXCR4 antagonists were shown to inhibit the migration of AML cells in response to CXCL12. In addition, such antagonists were shown to inhibit the survival and colony forming potential of AML cells and abrogate the protective effects of stromal cells on chemotherapy-induced apoptosis in AML cells. In vivo, using immune deficient mouse models, CXCR4 antagonists were found to induce the mobilization of AML cells and progenitor cells into the circulation and enhance anti leukemic effects of chemotherapy. The hypothesis that CXCL12/CXCR4 interactions contribute to the resistance of AML cells to signal transduction inhibitor- and chemotherapy-induced apoptosis is currently being tested in a series of Phase I/II studies in humans.

Acute myeloid leukemia (AML) with a complex karyotype (CK) has frequent alterations in TP53 and a very poor prognosis. We examined whether a prompt and simple fluorescence in situ hybridization (FISH) analysis for 17p13 deletion at diagnosis has a predictive value for response to therapy and overall survival in subgroups of AML. In 15 patients with a normal karyotype the TP53 FISH analysis was normal, whereas in 16 patients with CK 75% had only one copy of the TP53 allele. The deletion was also detected in 33% of six patients with monosomy or partial monosomy of chromosome 5, 7, 9, or 12. This loss of TP53 correlated significantly with a poor response to chemotherapy, and the median survival time of these patients was shorter. TP53 FISH analysis carried out at diagnosis has a predictive value with respect to chemotherapy response and can therefore facilitate a rapid decision on treatment strategies.

Poor prognosis of acute leukemia with current treatments is mainly due to the relapse of the disease following chemotherapy. In the last decade, an emerging concept has proposed that the leukemia stem cells (LSCs) and their interactions with the BM microenvironment are the major cause of the acute leukemia relapse. Adhesion to the stromal niche is crucial for LSCs as it directly supports self-renewal, proliferation, arrest of differentiation and protects from damaging chemo-agents. One of the key players in this crosstalk between leukemic cells and the BM stroma niche is the chemokine SDF-1. SDF-1 regulates the process of homing and engraftment of LSCs into the BM and inhibition of its receptor CXCR4 induces leukemic cell mobilization into the circulation. However, besides its chemotactic and adhesive functions, SDF-1 is also a pleiotropic cytokine that regulates leukemic cell proliferation as well as their program of differentiation. CXCR4 antagonists are used in combination with chemotherapy in preclinical and clinical studies, which demonstrate that blocking CXCR4 is a novel promising approach of therapy. In this review, we focus on the multifaceted SDF-1/CXCR4 axis in acute leukemia and discuss how targeting this pathway could provide potential interest to eradicate the LSCs.

Multiple myeloma is characterized by the malignant growth of immunoglobulin producing plasma cells, predominantly in the bone marrow. The effects of primary human mesenchymal stromal cells on the differentiation phenotype of multiple myeloma cells were studied by co-culture experiments. The incubation of multiple myeloma cells with bone marrow-derived mesenchymal stromal cells resulted in significant reduction of the expression of the predominant plasma cell differentiation markers CD38 and CD138, and cell surface immunoglobulin light chain. While the down-regulation of CD138 by stromal cells was completely dependent on their adhesive interactions with the multiple myeloma cells, interleukin-6 induced specific down-regulation of CD38. Mesenchymal stromal cells or their conditioned media inhibited the growth of multiple myeloma cell line, thereby reducing the overall amounts of secreted light chains. Analysis of primary multiple myeloma bone marrow samples reveled that the expression of CD38 on multiple myeloma cells was not affected by adhesive interactions. The ex vivo propagation of primary multiple myeloma cells resulted in significant increase in their differentiation markers. Overall, the data indicate that the bone marrow-derived mesenchymal stromal cells revert multiple myeloma cells to less differentiated phenotype by the combined activities of adhesive interactions and interleukin-6.

Stromal cell–derived factor-1 (SDF-1/CXCL12) and its receptor CXCR4 are implicated in the pathogenesis and prognosis of acute myelogenous leukemia (AML). Cellular microparticles, submicron vesicles shed from the plasma membrane of various cells, are also associated with human pathology. In the present study, we investigated the putative relationships between the SDF-1/CXCR4 axis and microparticles in AML. We detected CXCR4-expressing microparticles (CXCR4+ microparticles) in the peripheral blood and bone marrow plasma samples of normal donors and newly diagnosed adult AML patients. In samples from AML patients, levels of CXCR4+ microparticles and total SDF-1 were elevated compared with normal individuals. The majority of CXCR4+ microparticles in AML patients were CD45+, whereas in normal individuals, they were mostly CD41+. Importantly, we found a strong correlation between the levels of CXCR4+ microparticle and WBC count in the peripheral blood and bone marrow plasma obtained from the AML patients. Of interest, levels of functional, noncleaved SDF-1 were reduced in these patients compared with normal individuals and also strongly correlated with the WBC count. Furthermore, our data indicate NH2-terminal truncation of the CXCR4 molecule in the microparticles of AML patients. However, such microparticles were capable of transferring the CXCR4 molecule to AML-derived HL-60 cells, enhancing their migration to SDF-1 in vitro and increasing their homing to the bone marrow of irradiated NOD/SCID/β2mnull mice. The CXCR4 antagonist AMD3100 reduced these effects. Our findings suggest that functional CXCR4+ microparticles and SDF-1 are involved in the progression of AML. We propose that their levels are potentially valuable as an additional diagnostic AML variable. (Cancer Res 2006; 66(22): 11013-20)

Tumors contain a fraction of cancer stem cells that maintain the propagation of the disease. The CD34(+)CD38(-) cells, isolated from acute myeloid leukemia (AML), were shown to be enriched leukemic stem cells (LSC). We isolated the CD34(+)CD38(-) cell fraction from AML and compared their gene expression profiles to the CD34(+)CD38(+) cell fraction, using microarrays. We found 409 genes that were at least twofold over- or underexpressed between the two cell populations. These include underexpression of DNA repair, signal transduction and cell cycle genes, consistent with the relative quiescence of stem cells, and chromosomal aberrations and mutations of leukemic cells. Comparison of the LSC expression data to that of normal hematopoietic stem cells (HSC) revealed that 34% of the modulated genes are shared by both LSC and HSC, supporting the suggestion that the LSC originated within the HSC progenitors. We focused on the Notch pathway since Jagged-2, a Notch ligand was found to be overexpressed in the LSC samples. We show that DAPT, an inhibitor of gamma-secretase, a protease that is involved in Jagged and Notch signaling, inhibits LSC growth in colony formation assays. Identification of additional genes that regulate LSC self-renewal may provide new targets for therapy.

The role of the proteolytic enzyme elastase in motility and proliferation of leukemic human acute myeloblastic leukemia (AML) cells is currently unknown. We report a correlation between abnormally high levels of elastase in the blood of AML patients and the number of leukemic blast cells in the circulation. In AML cells, we observed expression of cell-surface elastase, which was regulated by the chemokine stromal cell-derived factor-1 (SDF-1). In vitro inhibition of elastase prevented SDF-1-induced cell polarization, podia formation, and reduced migration of human AML cells as well as their adhesion. Elastase inhibition also significantly impaired in vivo homing of most human AML cells to the bone marrow (BM) of nonobese diabetic-severe combined immunodeficient (NOD/SCID)/beta-2 microglobulin knock-out (B2mnull) mice that underwent transplantation. Moreover, in vitro proliferation of AML cells was elastase dependent. In contrast, treatment with elastase inhibitor enhanced the proliferation rate of human cord blood CD34+ cells, including primitive CD34+/CD38- cells, and their in vivo homing. Finally, NOD/SCID mice previously engrafted with human AML cells and treated with elastase inhibitor had significantly reduced egress of leukemic cells into the circulation. Taken together, our data demonstrate that human AML cells constitutively secrete and express SDF-1-dependent cell-surface elastase, which regulates their migration and proliferation. (Blood. 2005;106:2120-2127)

Mutations of transcription factors are associated with the pathogenesis of cancer. Genomic DNA from 381 cancers and cell lines representing leukemias, lymphomas and a variety of solid tumors were examined for mutations of genes coding for the C/EBP-beta, C/EBP-alpha, PU.1, and AML1 transcription factors using single strand conformation polymorphism (SSCP) and direct DNA sequencing. Mutation of C/EBP-beta (a chronic myelogenous leukemia cell line, Kcl22) and C/EBP-delta (a Burkitt's lymphoma cell line, Raji) were found. Interestingly, the sample with a C/EBP-beta alterations had two missense (P236L and G252A) and two silent mutations in a highly conserved region of the gene. The C/EBP-delta alteration in Raji was a missense mutation (A177G). These findings suggest that mutations of the C/EBP-beta, C/EBP-delta, PU.1, and AML1 rarely contribute to the development of hematopoietic or solid cancers.

Checkpoint genes code for a family of proteins which sense DNA damage in eukaryotic cells. They play an important role in the control of the cell cycle. The human CHK2 is a homolog of the yeast G2 checkpoint kinases known as CDS1 and RAD53. The CHK2 may be a tumor suppressor gene because it was found to be mutated in some individuals with the Li–Fraumeni syndrome. These cases had a normal, non-mutated p53 gene. We performed a mutational analysis of the CHK2 gene using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) in 41 bone marrow samples from individuals with myelodysplastic syndrome (MDS) and 41 samples of acute myeloid leukemias (AML). We found a novel G to C transversion resulting in a change from Ala to Gly at codon 507 of CHK2 in one MDS sample, but normal cells from this individual did not have the abnormality. In addition, we demonstrated a previously described polymorphism at codon 84 (A to G at nucleotide 252) of exon 1 of CHK2 in three of 41 MDS and three of 41 AML patients. The presence of a CHK2 mutation in MDS highlights the importance of alterations of cell cycle checkpoint genes in this disease.

The CHK2 gene encodes a protein kinase that is important for the regulation of cell cycle arrest after DNA damage. CHK2 acts downstream of ataxia teleangiecstasia mutated (ATM), modulates the function of p53 and may help mediate cell cycle arrest at G2/M by phosphorylation of Cdc25C. Recently, the human homolog of the checkpoint kinase Cds1 (CHK2) has been suggested to be a tumor suppressor gene. Heterozygous germline mutations have been reported in Li-Fraumeni syndrome (LFS), a highly penetrant familial cancer phenotype, and in sporadic colon cancer. LFS is associated with the development of lymphoid malignancies, especially childhood ALL. Therefore, we analyzed the DNA from 143 lymphoid malignancies to determine whether they had mutations of the CHK2 gene. The 14 exons of CHK2 were studied by polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) and sequencing of aberrantly migrating bands. One missense mutation changing serine to phenylalanine (codon 428) in an evolutionarily highly conserved domain was found in a non-Hodgkin's aggressive lymphoma. Another point mutation in the non-coding region was identified in one of adult T-cell leukemias (ATL) samples. This result suggests that mutation of the CHK2 gene may rarely be involved in the development of selected lymphomas.

Recent studies have reevaluated whether gemtuzumab ozogamicin (GO) improves the outcome of acute myeloid leukemia (AML) in elderly patients. Over 5 years, we treated 16 elderly patients with AML with GO and cytarabine. A high response rate, prolonged survival, and low toxicity were observed in the favorable and intermediate-I genetic groups of AML. Our study raises the issue about the optimal protocol for these patients. Background: The benefit of gemtuzumab ozogamicin (GO) in combination with chemotherapy as frontline therapy in patients with acute myeloid leukemia (AML) is still debated. Patients and Methods: We evaluated the safety and efficacy of low-dose GO with cytarabine in elderly patients with newly diagnosed AML. Over the past 5 years, we have treated 16 elderly patients with AML (64-82 years) with GO (3 mg/m 2 ) followed by continuous infusion of cytarabine (100 mg/m 2 ) for 7 days. Results: Complete remission (CR) was achieved in 68.8% of patients; however, this was true only in patients in the favorable or intermediate-I cytogenetic risk groups. Of the 12 patients with AML in the favorable and intermediate-I genetic groups, 11 (91.7%) achieved CR. By comparison, of all 4 patients in the intermediate-II or adverse genetic groups, none of the patients achieved CR (P .003). The median disease-free survival and overall survival (OS) was 10.9 and 18.8 months, respectively, for patients who achieved CR. The estimated median survival was 15 months in the favorable and intermediate-I cytogenetic groups and only 4.4 months in the intermediate-II and unfavorable risk groups (P .008). The toxicity profile was also manageable in patients with AML who were mainly older than 70 years with good performance status (PS). The 8-week mortality rate was 6.25%, which is relatively low in this high-risk group of patients. These data are in line with results from 2 randomized trials suggesting that the addition of low-dose GO should be further investigated to reevaluate its role in selected elderly patients with AML and raises the issue of the optimal protocol.

The outcome and quality of life of chronic myeloid leukemia (CML) patients has remarkably changed with the treatment of tyrosine kinase inhibitors (TKIs). Currently, hematopoietic stem cell transplantation (HSCT) is considered mainly as a third line salvage therapy in cases of TKIs resistance or intolerance. Here we describe a patient with chronic phase CML who developed both resistance and late occurrence of s severe thrombocytopenia on first and second generation TKIs and eventually underwent HSCT. Although the mechanism of the myelosuppression is not fully understood, we showed for the first time the development of dose dependent platelet antibodies in the presence of TKIs, suggesting the possibility of TKIs induced thrombocytopenia. Our case emphasizes that late development of severe myelosuppression during imatinib treatment is probably an important indication for consideration of early HSCT.

Xenografting of human blood malignancies to immunodeficient SCID mice is a powerful research tool. We evaluate here whether the immunodeficient turkey embryo can also serve as a xenograft host for human blood malignancies. Human leukemia, lymphoma and myeloma lines engrafted robustly into medullary and extramedullary tissues of turkey embryos as detected by PCR, FACS and histology in 8–10 days. Four of eleven patient AML samples also engrafted the bone marrow. Grafts of two lines responded to chemotherapy with doxorubicin. The turkey embryo therefore has the potential to be a complementary xenograft model for the study of human blood malignancies.

Resistance to chemotherapy and subsequent relapse is the most challenging issue in the treatment of patients with Acute Myeloid Leukemia (AML). However, the underlying mechanisms still remain incompletely understood. Here we report that loss of the histone methyltransferase EZH2 and subsequent reduction of H3K27 trimethylation contribute to chemoresistance in AML. In Myelodysplastic Syndrome (MDS) and Myeloproliferative Neoplasms (MPN) EZH2 is often inactivated due to mutations which is associated with poor prognosis. By use of quantitative PCR and immunohistochemistry we show that a decrease of EZH2 mRNA and protein also correlated with a poor prognosis of AML patients indicating a tumor suppressor role of EZH2 in AML. EZH2 is located on chromosome 7q36.1 and it is not yet fully clear whether EZH2 expression is affected in MDS and AML patients with del(7)/del(7q) who are largely refractory to chemotherapy and have a poor prognosis. We found EZH2 levels to be reduced in del(7)/del(7q) AML patients as determined by Western Blot. Notably, the reduction of EZH2 protein levels via treatment with H3K27 methyltransferase inhibitors or lentiviral knockdown was sufficient to induce chemoresistance of Normal Karyotype (NK)- AML blasts and cell lines in vitro and in a xenograft mouse model. Furthermore, we observed that EZH2 loss occurred during the acquisition of drug resistance in a Tyrosine Kinase Inhibitor- and Cytarabine (AraC)- resistant AML cell line. Pharmacological inhibition of CDK1 and treatment with the proteasome inhibitor Bortezomib, respectively, increased EZH2 protein and restored drug sensitivity. Functionally, the loss of EZH2 directly induced upregulation of HOX genes, suggesting a stem-cell-like signature to be associated with the resistance phenotype, which could be reverted by Bortezomib treatment. To evaluate the potential of Bortezomib to affect EZH2 levels in patient blasts we treated primary NK-AML blasts collected at diagnosis ex vivo with Bortezomib. In almost all samples Bortezomib treatment induced cytotoxic effects. In 5 out of 10 patients the EZH2 protein level could be increased by Bortezomib treatment. We furthermore examined the sensitivity of patient samples to AraC, Bortezomib or combined treatment. Notably, for those patients with increased EZH2 levels after Bortezomib exposure we found a significantly decreased cell survival for the combined treatment compared to single-agent treatment. Our data strongly suggest that restoration of EZH2 protein levels e.g. via proteasome inhibitors and thereby restoration of EZH2 function might be a novel promising approach to increase therapy response in AML. Citation Format: Stefanie Göllner, Shuchi Agrawal-Singh, Tino Schenk, Hans-Ulrich Klein, Christian Rohde, Tim Sauer, Mads Lerdrup, Sigal Tavor, Friedrich Stölzel, Gerhard Ehninger, Gabriele Köhler, Martin Dugas, Arthur Zelent, Christian Thiede, Wolfgang E. Berdel, Klaus Hansen, Carsten Müller-Tidow. Loss of the histone methyltransferase EZH2 induces chemoresistance in acute myeloid leukemia (AML). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4430. doi:10.1158/1538-7445.AM2015-4430

Introduction: Ruxolitinib has been shown in two randomized clinical trials (RCTs) to be effective in alleviating systemic symptoms and effecting a reduction in spleen size in patients with myelofibrosis (MF). However, JAK2 allele burden is not significantly impacted by this drug and there is no consistent salutary effect on marrow fibrosis or definite improvement in overall survival. Currently, the goals of Ruxolitinib treatment remain palliative. We sought to determine the efficacy and tolerability of Ruxolitinib in a cohort of unselected patients with MF treated in routine clinical practice. Methods: MF patients treated with Ruxolitinib for at least 3 months in 13 participating centers that are members of the Israel Myeloproliferative Neoplasm (MPN) Working Group were identified. The following demographic and clinical data were analyzed: the form of MF [primary (PMF), post polycythemic myelofibrosis (PPVMF), post essential thrombocytosis myelofibrosis (PETMF)], duration of MPN, indication for treatment, initial dose, dose reduction, hematologic toxicity, response to therapy and withdrawal of treatment. Results: One hundred and two patients from 13 centers that began Ruxolitinib between January 2012 and April 2014 were identified. Ninety three patients who were treated for more than 3 months were included in the analysis. Median age at diagnosis was 59 years (range 25-84), 57 % were males. PMF was the diagnosis in 44 patients (47.3%), PPVMF in 29 (31.2%) and PETMF in 17 (18.3%). The median duration of disease was 5 years (range 3 months-35 years) for the entire cohort. Median age at Ruxolitinib initiation was 67 years (range 32-84). Seventy two (78.3%) patients received cytoreductive therapies for MF prior to Ruxolitinib. Indications for treatment were constitutional symptoms only in 14 patients (15%), symptomatic splenomegaly only in 6 patients (6%) and both in 71 patients(76%). Two patients received Ruxolitinib for other indications (non-constitutional symptoms and refractory thrombocytosis). The median initial dose of Ruxolitinib was 30 mg per day (range 10-40mg). Median duration of Ruxolitinib therapy was 11 months (range 3-31 months). Eighty two patients (88.2%) responded to therapy, 76 (84.4%) patients had improvement in constitutional symptoms and 60 patients (70.6%) had reduction in spleen length. While on Ruxolitinib, 60 patients (64%) had a nadir hemoglobin level of less than 10g/dL, 43 patients (46%) had a nadir platelet count of less than 100 x 109/µL and in 12 of them (12.9%) a platelet nadir was less than 50 x 109/µL. Twenty one patients (22.6%) needed packed red blood cell (PRBC) transfusion in the 2 months preceding Ruxolitinib initiation and they received a median of 2.5 units (range 1-8), while 27 patients (29.1%) needed PRBC transfusion in the first 2 months after starting treatment and they received a median of 4 units (range 1-8). Thirty five patients required dose reduction of Ruxolitinib and 14 (15.2%) discontinued their therapy. Univariate analysis revealed that response to Ruxolitinib occurred in patients with a lower white blood cell (WBC) count (median 9.7 x 103/µL vs 21.5 x 103/µL; P=0.033), a greater degree of splenomegaly (median 12 vs 4 cm below costal margin; P=0.001) and hepatomegaly (median 4 vs 0 cm below costal margin; P=0.011). In multivariate analysis, the degree of splenomegaly was found to be predictive of response to treatment (odds ratio=1.263, p=0.03 and 95% CI=1.08-1.476) while there was a trend to improved response in patients with a lower WBC (p=0.074). Conclusions: The present analysis of a cohort of MF patients treated with Ruxolitinib in routine clinical practice has demonstrated the efficacy and tolerability of this drug outside of a highly monitored RCT setting. Data of this sort are currently sparse, and emanate mainly from single-center reports. Our study performed in 13 academic and community hospitals provides real-life evaluation of the utility of Ruxolitinib and factors associated with response to treatment. Disclosures No relevant conflicts of interest to declare.

The chemokine stromal cell-derived factor-1 (SDF-1) and its receptor CXCR4 participate in the retention of normal hematopoietic stem cells within the bone marrow (BM) and their release into the circulation. Homing and engraftment of human stem cells in immunodeficient mice are dependent on cell surface CXCR4 expression and the production of BM SDF-1, which acts also as a survival factor for both human and murine stem cells. However, the role of SDF-1/CXCR4 interactions in the control of human acute myelogenous leukemia (AML) cell trafficking and disease progression is poorly understood. In this study, we report that although some AML cells do not express surface CXCR4, all AML cells tested express internal CXCR4 and SDF-1. Culture of AML cells with SDF-1 promoted their survival, whereas addition of neutralizing CXCR4 antibodies, SDF-1 antibodies, or AMD3100 significantly decreased it. Pretreatment of primary human AML cells with neutralizing CXCR4 antibodies blocked their homing into the BM and spleen of transplanted NOD/SCID/B2mnull mice. Furthermore, weekly administrations of antihuman CXCR4 to mice previously engrafted with primary AML cells led to a dramatic decrease in the levels of human AML cells in the BM, blood, and spleen in a dose- and time-dependent manner. Interestingly, the same treatment did not affect significantly the levels of normal human progenitors engrafted into NOD/SCID mice. Taken together, our findings demonstrated the importance of the SDF-1/CXCR4 axis in the regulation of in vivo motility and development of human AML stem cells and identified CXCR4 neutralization as a potential treatment for AML.

The transcription factor C/EBPalpha plays a critical role in the process of granulocytic differentiation. Recently, mutations that abrogated transcriptional activation of C/EBPalpha were detected in acute myeloid leukemia patient samples. Moreover, the progression of chronic myelogenous leukemia (CML) to blast crisis in patients was correlated with down-modulation of C/EBPalpha. The KCL22 cell line, derived from BCR-ABL+ CML in blast crisis, expressed wild-type C/EBPepsilon protein but not a functional C/EBPalpha, -beta, and -gamma. Restoration of C/EBPalpha expression in KCL22 cells triggered a profound proliferative arrest, a block in the G2/M phase of the cell cycle and a gradual increase in apoptosis. Within 3 days of inducing expression of C/EBPalpha, a remarkable neutrophilic differentiation of the KCL22 blast cells occurred as shown by morphologic changes, induction of expression of CD11b, primary, secondary, and tertiary granule proteins, and granulocyte colony-stimulating factor receptor. Using high density oligonucleotide microarrays, the gene expression profile of KCL22 cells stably transfected with C/EBPalpha was compared with that of empty vector, and we identified genes not previously known to be regulated by C/EBPalpha. These included the up-regulation of those genes important for regulation of hematopoietic stem cell homing, granulocytic differentiation, and cell cycle, whereas down-regulation occurred for genes coding for signaling molecules and transcription factors that are implicated in regulation of proliferation and differentiation of hematopoietic cells. Our study showed that restoration of C/EBPalpha expression in BCR-ABL+ leukemic cells in blast crisis is sufficient for rapid neutrophil differentiation suggesting a potential therapeutic role for ectopic transfer of C/EBPalpha in acute phase of CML.

Members of the CCAAT/enhancer-binding protein (C/EBP) family are involved in the regulation of cellular differentiation and function of many tissues. Unlike the other members of the family, C/EBP epsilon expression is restricted to granulocytes, macrophages, and lymphocytes. C/EBP epsilon is highly conserved between human and rodents and is essential for terminal granulopoiesis in both species. To study the role that C/EBP epsilon plays in macrophages, wild-type and C/EBP epsilon-deficient (-/-) murine macrophages obtained from thioglycollate-elicited peritoneal lavages and differentiated bone marrow cells were compared. Although macrophage development occurred in both types of mice, the C/EBP epsilon -/- cells had a lower expression of macrophage markers and a morphologic and ultrastructural appearance of immaturity. Phagocytic function, measured by calculating the percentage of internalized opsonized fluorescein isothiocyanate (FITC)-labeled yeast, was significantly impaired in the C/EBP epsilon -/- macrophages compared with their wild-type counterparts. Furthermore, the differential expression of 26 macrophage-specific genes between wild-type and C/EBP-/- mice was analyzed. A subset of genes involved in differentiation, immune, and inflammatory responses was found down-regulated in the C/EBP-/- macrophages. Taken together, this study implicates the C/EBP epsilon gene as an important transcription factor required for normal function and development of macrophages.

Checkpoint genes, activated in response to DNA damage and other stresses, are frequently targeted for alteration in cancer. Checkpoint kinase 2 (CHK2, CDS1, RAD53) is activated by ataxia telangiectasia mutated (ATM) in response to gamma irradiation. Activated CHK2 stabilizes TP53, and acts on other cell cycle and stress regulators. These findings place CHK2 in the middle of a pathway frequently targeted in cancer. Because of this, and the observation that CHK2 mutations are inherited in some Li-Fraumeni cancer syndrome families, we decided to examine the role of CHK2 mutations in sporadic cancers. Exploiting the genomic sequence of chromosome 22, we looked for mutations in the exons and intron junctions of the CHK2 gene in DNA samples from 170 patients (57 osteosarcomas, 25 other sarcomas, 35 nonsmall-cell lung, 20 ovarian, and 33 breast cancers). Missense mutations affecting the forkhead and kinase domains were detected in four osteosarcomas and in one ovarian and one lung cancer. These findings of CHK2 gene mutations are consistent with osteosarcoma being a defining tumor of Li-Fraumeni syndrome. The occurrence of CHK2 mutations in sporadic cancers emphasizes the importance of the stress pathway which includes TP53.

Thrombotic thrombocytopenic purpura (TTP) is an uncommon disease of an unknown etiology, characterized by consumptive thrombocytopenia, microangiopathic hemolytic anemia, fever and acute thrombotic complications, especially within the cerebral circulation. Although anti-endothelial cell antibodies (AECA) have occasionally been shown to be present in TTP, their role in the pathogenesis of the disease has never been ascertained. In the current study we demonstrated the pathogenic activity of affinity-purified anti-endothelial cell F(ab)2 antibodies (AECA/TTP) from four consecutive patients with active TTP. These AECA/TTP bound to and activated only microvascular endothelial cells (EC) and not large vessel EC. The specificity of AECA/TTP binding to microvascular EC was confirmed by competition assay employing membranes derived from small and large vessels EC. Activation included enhanced IL-6 and von Willebrand factor release from the EC followed by increased expression of adhesion molecules P-selectin, E-selectin and vascular cell adhesion molecule-1 on the EC, as evaluated by ELISA. Increased expression of adhesion molecules was followed by an increase in monocyte adhesion to EC. The level of soluble thrombomodulin (TM) also increased in the culture medium of activated microvascular EC upon exposure to AECA/TTP antibodies and was directly correlated to a decrease in cell-associated TM. Our data suggest that AECA/TTP directed against microvascular EC could play a pathogenic role in the development of endothelial injury in TTP that leads to thrombosis.

Multiple myeloma (MM) is an incurable hematopoietic malignancy of terminally differentiated B cells, monoclonal plasma cells, which accumulate and grow predominantly in the bone marrow (BM). It is ...

Histone modifications play a crucial role in the regulation of gene expression by activating or inactivating transcription. The Polycomb Group Protein Enhancer of Zeste Homologue 2 (EZH2) mediates trimethylation of histone H3K27, thereby inducing gene silencing. Overexpression of EZH2 has been reported to be associated with metastases and cancer progression in solid tumors like breast cancer or prostate cancer. However, loss of function mutations or deletions of EZH2 occur in myeloid malignancies and T-ALL. These mutations result in a poor prognosis. The aim of this study was to analyze the relevance of histone modifications for therapy resistance in AML. FLT3-ITD positive MV4-11 leukemic cells that were continuously cultured in media containing the kinase inhibitor PKC412 became resistant not only to PKC412 but also to standard chemotherapeutics Cytarabin (AraC) and Daunorubicin. Western blot analysis identified an almost complete loss of H3K27me3 in resistant MV4-11 cells (MV4-11R). This was accompanied by loss of EZH2 protein in the MV4-11R compared to the sensitive MV4-11. To test for acquisition of drug resistance due to reduced H3K27me3 levels, lentiviral knock-down (KD) of EZH2 was performed in the sensitive MV4-11 leading to diminished H3K27me3 levels. Knock-down cells showed resistance to the apoptosis-inducing effects of PKC412 compared to scrambled controls. Furthermore, resistance to standard chemotherapeutics AraC and Daunorubicin could be also observed in MV4-11 KD cells compared to control. To verify whether diminished levels of H3K27me3 can cause a more general, FLT3-ITD-independent drug resistance, knock-down of EZH2 was performed in FLT3-WT AML cell lines HL60, Kasumi-1 and ML-1. Again, this led to resistance to the standard chemotherapeutics AraC and Daunorubicin. In order to investigate the regulation of EZH2 in MV4-11R, promoter methylation and microRNA expression analysis was performed revealing no regulation of EZH2 expression via both mechanisms. Instead, the reduction of EZH2 protein expression was depended on posttranslational mechanisms that could be counteracted by CDK1-inhibitors. CDK1-inhibitors restored EZH2 protein and H3K27 trimethylation levels as well as drug sensitivity. By analyzing EZH2 mRNA expression of 220 primary diagnosed AML patients, a trend towards low EZH2 mRNA expression and poor overall as well as relapse free survival could be demonstrated. Thus, EZH2- and H3K27me3 protein expression were also analyzed by immunohistochemistry in bone marrow biopsies from AML patients (N=126). H3K27me3 and EZH2 protein expression correlated closely (r= 0.9, p Taken together, these data indicate that loss of EZH2 expression and reduction of H3K27me3 levels induce widespread therapy resistance in AML and associate with a poor prognosis in AML patients. Disclosures: No relevant conflicts of interest to declare.

Background The chemokine CXCL12 and its receptor CXCR4 are key players in mediating the interactions between the bone marrow (BM) microenvironment and Acute Myeloid Leukemia (AML) cells. CXCL12, which is constitutively secreted from the BM stroma and AML cells, is critical for the survival and retention of AML cells within the BM. CXCR4 expression is associated with poor prognosis in AML patients with or without a mutated FLT3 gene. Antagonists to CXCR4 inhibit migration of AML cells, induce mobilization of AML cells into the circulation and enhance anti-leukemic effects of chemotherapy in mice models. The hypothesis that CXCL12/CXCR4 interactions contribute to the resistance of AML cells to signal transduction inhibitor, and chemotherapy-induced apoptosis, is currently being tested in a series of clinical trials in humans. Methods In the present study, the effect of the high affinity CXCR4 antagonist BL-8040 (BKT140) and AMD3100 (Mozobil) alone and in combination with ARA-C or the FLT-3 inhibitor AC220 on the survival and proliferation of AML cells in-vitro was examined. In the in-vitro study HL60 (FLT3-WT), MV4-11 (FLT3-ITD) cell lines and human primary AML cells from patients with FLT3-ITD mutations and FLT3-WT genewere used. Cells were incubated for 48 hrs in the presence of BL-8040 (8µM-20µM), AMD3100 (20µM), ARA-C (10-200 ng/ml) and AC220 (0.5-50nM). The level of viable cells, percentage of apoptosis and cell cycle were evaluated by FACS using propidium iodide and 7-AAD. In-vivo, we used NOD scid gamma (NSG) mice engrafted with human primary AML blasts and explored the effects of single injection of BL-8040 on the mobilization and survival of the blasts in the blood and the BM of the engrafted mice. Results In-vitro, treatment of MV4-11 cells (FLT3-ITD) with BL-8040, unlike treatment with AMD3100, directly inhibited cell growth by 35% and increased cell death by 39%. Furthermore, in-vitro, treatment of primary AML cells (FLT3-ITD) with BL-8040 directly inhibited cell growth by 28-47% and increased cell death by 75-100%. A combination of BL-8040 with AC220 or ARA-C further increased the apoptotic effect of these agents achieving a 96% reduction in cell viability and inducing cell death by 70- 90% of AML cells. When we studied the in-vitro effect of these agents on FLT3-WT cells (HL-60 cell line and primary AML cells), we found that BL-8040 inhibits cell growth by 16-50%. Unlike the FLT3-ITD cells, in the FLT3-WTcells we did not observe additive effects on cell growth for the combined treatments of BL-8040 with AC220. The combined treatment of BL-8040 with ARA-C was found to further increase the percentage of AML cell death. Moreover, BL-8040 decreases the percent of cycling cells by reducing the number of cells in G2/M+S phase while increasing the number of apoptotic cells in sub-G0 phase. It is interesting to mention that the migration of all tested AML cells toward CXCL12 was entirely inhibited by BL-8040. In-vivo, we found that a single injection of BL-8040 (100μg/mice) into NSG mice engrafted with human AML cells, induces rapid mobilization of AML cells to the periphery within 4 hrs after injection (an 8 fold increase from the control). When mice were administered with 5 consecutive injections of BL-8040 (400μg/mice), a reduction in the number of AML blasts in the blood was observed (40-60% reduction) with induction of AML cell apoptosis within the BM and in the blood by 10-20-fold, compared to the control. Conclusions The CXCR4 antagonist BL-8040 was found to rapidly and efficiently induces cell death of AML cells both in-vitro and in-vivo. These results suggest potential therapeutic advantages of BL-8040 in both FLT3-positive and negative AML patients by targeting not only AML anchorage in the BM but AML survival as well. Furthermore, it could provide a rational basis for BL-8040 therapy in combination with ARA-C and the FLT3 inhibitor AC220. Disclosures: Eizenberg: Biokine: Employment. Pereg:BioLineRx LTD: Employment. Klapper:BioLineRx LTD: Employment. Abraham:Biokine: Employment.

Tavor et al. [(1)][1] investigated the role of CXCR4 in the migration and development of human acute myelogenous leukemia (AML) stem cells in NOD/severe combined immunodeficient (SCID) mice. They reported that human AML cells home to the murine bone marrow (BM) and spleen in an SDF-1/CXCR4-dependent

Abstract 2617 Poster Board II-593 In recent years, a number of studies emphasized AML with complex aberrant karyotype as a distinct biological entity which is characterized by a unique gene expression pattern, with very frequent alteration in TP53 and a median overall survival (OS) of less than 6 months. However, the definition of a complex karyotype (CK) or whether monosomal karyotype (MK) provides a better prognostic prediction than CK is not well established. In the present study, we examined whether a prompt and simple Fluorescence in situ hybridization (FISH) test for TP53 deletion (presence or absence of 17p13) at diagnosis, has a predictive value for response to therapy and overall survival in AML patients (pts) with CK or chromosomal deletions (either complete or partial of any other chromosome). Between 2000 and 2009 we analyzed 38 patients with newly diagnosed AML, with age ranged from 18 to 82 years (median 52 years). On cytogenetic analysis from bone marrow at diagnosis, 16 (42%) pts had a CK (≥3 chromosome abnormalities), 16 pts (42%) had a normal karyotype (NK) and 6 (16%) pts had other monosomy or deletion of any chromosomal region. Seven patients had a post myelodysplastic syndrome AML and 1 post therapy for a prior malignancy. For induction treatment patients received idarubicine, 12 mg/m2/d (age 18– 55y) or mitoxantron 10 mg/m2/d intravenous (IV) on days 1-3 (age 55–68y) and cytarabine, 100mg/m2/d by continuous IV infusion on days 1 through 7. Pts entering complete remission (CR) received three courses of consolidation with high dose cytarabine. Patients with a histocompatibale donor, which did not received CR or relapsed, were allografted. Elderly patients, 69–82 years old, were treated with one induction chemotherapy which included Mylotarg (3mg/m2/2h on day 1) plus cytarabine (100 mg/m2/24h on days 2-8). Patients at this group of age did not received consolidation therapy. Of the16 pts with CK AML, 14 were treated with chemotherapy but only 3 pts (21%) achieved CR, 4 pts died during induction therapy. By comparison, of the 16 pts with NK AML, 15 pts were treated with chemotherapy, 11 pts achieved CR (73%), 3 pts died during induction therapy. Of the 6 AML pts with monosomy or deletion in cytogenetic analysis, 3 pts (50%) achieved CR (p =0.0067). In all NK AML patients examined the TP53 FISH was normal (mean 4% of cells examined), where as in patient with CK 75% of pts had TP53 deletion (mean 51%), and in the group with chromosomal deletion 50% of pts showed a loss of one TP53 (mean 18%) p=0.0016. Of note, in 13 pts (34%) with TP53 deletion by FISH analysis, cytogenetic analysis found no anomaly of 17p chromosome. In a stepwise logistic regression model FISH groups significantly entered the model in first step, and no other variable did (p=0.0056, C=0.833). In pts in which two copies of TP53 were found in FISH analysis ('10%) the median survival time is 440 days, whereas in pts with TP53 deletion was detected the median survival time was 189 days. The OS in pts with deletion of TP53 was significantly shorter as compared to pts that TP53 deletion was not found (p=0.025). However, a multivariate analysis including age, karyotype, deletion of TP53 and allogeneic transplantation showed that only CK and allogeneic transplantation are independent prognostic factors in this analysis. In conclusion, TP53 status by FISH at diagnosis is important due to the following findings: The test is sensitive, rapid and simple. 2. TP53 deletions are frequent in AML with CK and tend to segregate with additional chromosomal deletions. TP53 status at diagnosis has a predictive value with respect to chemotherapy response and OS in AML pts with CK and chromosomal deletions. Disclosures: No relevant conflicts of interest to declare.

The chemokine stromal cell-derived factor-1 (SDF-1) and its receptor, CXCR4, participate in the retention of AML cells within the bone marrow microenvironment and their release into the circulation. We previously showed that the expression of cell surface elastase in AML cells is dependent on the SDF-1/CXCR4 axis, giving a first evidence for the association between the SDF-1 and elastase pathways. In the present study, we hypothesized that inhibition of the SDF-1/CXCR4 axis or elastase may regulate similar pathways and genes in AML cells. In order to test our hypothesis, we compared gene expression profiles of U937 AML cells that express high level of membrane CXCR4, treated with neutralizing CXCR4 mAb or elastase inhibitor (EI), with non-treated cells, by employing gene-array technology. Unsupervised hierarchical clustering analysis demonstrated similar gene expression profiles of anti-CXCR4 antibody or EI treated cells, as compared to control. Analysis of the 364 genes that were differentially expressed in both anti CXCR4 mAb or EI treated cells under both treatment agents identified genes that correspond to the transcriptional profiles of differentiated myeloid cells in a significantly high prevalence. Quantitative Real-time RT-PCR for 4 selected genes C/EBPe, FLT3, HOXA9 and G-CSFR, demonstrated gene expression profiles identical to the microarray expression profiles. Given thus, we further analyzed the effect of CXCR4 inhibition on AML cell growth and differentiation using the antagonist AMD3100. AMD3100 arrested proliferation in five AML cell lines as was suggested by proliferation assay, cell cycle and caspase 3 activity analyses. Moreover, cell differentiation was demonstrated by: Modifications in cell morphology, Increased expression of myeloid differentiation antigens Acquisition of the ability to produce oxidative bursts An increase in DNA ploidy in megakaryoblasts. Our study defines a new role for the SDF-1/CXCR4 axis in the regulation of leukemic cell survival and differentiation.

Elastase, along with other azurophil granule proteins like proteinase 3 regulates normal and leukemic granulopoiesis in an un-defined mechanism. We have recently showed that human acute myeloid leukemic (AML) cells constitutively express and secrete stromal derived factor 1 (SDF-1) dependent cell surface elastase, which regulates their migration and proliferation. To elucidate the molecular events and genes regulated by elastase and SDF-1/CXCR4 axis in AML cells, we examined gene expression of U937 AML cell line treated with neutralizing anti-CXCR4 Abs or elastase inhibitor (EI) compared to untreated cells, using DNA microarray technology. Unsupervised hierarchical clustering analysis showed very similar gene expression profiles of EI and anti CXCR4 Abs treated cells as compared to control. 230 of 8400 genes interrogated were repressed, and 164 were induced after culturing AML cells in the presence of EI or anti CXCR4 Abs at different time points as compared to untreated cells. Inhibition of elastase or CXCR4 was accompanied by down regulation of the transcripts of primary granule proteins. Functional classification of elastase or SDF-1/CXCR4 axis regulated genes revealed downregulation of HOXA9, HOXA10, ETS2, as well as other transcription factors that are over expressed in AML and are important for the development of leukemia. Whereas, transcriptional factors and regulators known to be induced during myeloid differentiation like C/EBPε, ID1, RUNX3 and HHEX were up-regulated in treated cells. Expression patterns of apoptosis genes indicated decline in death control by the p53 dependent pathway and a more prominent control by mitochondrial mediated apoptotic pathway like bcl2 related genes. In addition, receptors for interleukins, growth factors (G-CSFR and GM-CSF), complement component (C1QR1) were upregulated in the treated cells. In contrast, FLT-3, a growth factor receptor stimulating growth of early progenitor cells and AML blasts, was down regulated in AML cell treated with EI or anti CXCR4 Abs. These data were confirmed by real time PCR for selected marker genes of granulocytic differentiation. Interestingly, many of the differentially expressed genes were common to the transcriptional program of normal terminal granulocytic differentiation (Theilgaard-Monch & Borregarrd 2005. Blood 105:1785) suggesting that inhibition of elastase may induce differentiation in AML cells. Thus we further analyzed the effect of elastase inhibition on AML cell differentiation and growth. Treatment of HL60 AML cell line with EI triggered a proliferative arrest, apoptosis and mimicked terminal granulocytic differentiation, including morphologic changes, increased CD11b expression, and the ability to produce oxidative bursts. In summary, our study showed that inhibition of elastase or SDF-1/CXCR4 axis in AML cells affects similar pathways related to differentiation and malignant transformation, implying a critical role for those molecules in regulating leukemic development. Repression of elastase decreases proliferation and induces differentiation of AML cells, suggesting a potential new therapeutic approach for AML.

Stromal cell-derived factor-1 (SDF-1/CXCL12) and its receptor CXCR4 are implicated in the pathogenesis and prognosis of AML. Cellular microparticles (MPs), the submicron vesicles shed from the plasma membrane of various circulating cells, are associated with numerous human disorders. In the present study, we studied the putative relationships between CXCR4/SDF-1 axis and MPs in AML. We detected CXCR4 expressing MPs (CXCR4+MPs) in the peripheral blood and bone marrow plasma samples of normal donors (n=24) and newly diagnosed adult AML patients (n=26). The majority of CXCR4+MPs in AML patients were CD45+ whereas in normal individuals they were mostly CD41+. In samples from AML patients, the levels of CXCR4+MPs and total SDF-1 were significantly elevated as compared to normal individuals. Importantly, we found a strong correlation between the levels of CXCR4+MP and white blood cell (WBC) counts in the peripheral blood and bone marrow plasma obtained from the AML patients. Of interest, functional, non-cleaved SDF-1 levels were reduced in these patients compared to normal individuals, and also strongly correlated with the WBC counts. Furthermore, our data indicate N-terminal truncation of the CXCR4 molecule in the MPs of AML patients. This was found also in MPs obtained from the conditioned media of normal human CD34+ progenitors lentiviarlly transduced with CXCR4 vector in vitro. Appearance of MPs possessing N-terminally truncated CXCR4 in AML patients is likely to be dependent on proteolytic enzymes, such as elastase, which was elevated. However, MPs isolated from AML patients were capable of transferring functional CXCR4 molecule to the AML-derived HL-60 cells, enhancing their migration to SDF-1 in vitro and increasing their homing to the bone marrow of irradiated NOD/SCID mice. The CXCR4 antagonist AMD3100 reduced the increased migration and homing of MP treated HL-60 cells. Taken together, these findings suggest that functional CXCR4+MPs and SDF-1 are involved in the progression of AML. We propose that their levels are potentially valuable as an additional diagnostic AML parameter. Moreover, our findings suggest also the need for CXCR4- and SDF-1-target therapeutic approaches, clinically relevant in AML in the near future.

Neutrophil -or leukocyte- elastase is a serine protease stored in azurophilic granules of myeloid cells and is released upon activation and degranulation. Elastase degrades a number of extracellular matrix proteins as well as various cytokines and their receptors and may facilitate neutrophil motility. Lately, others and we have shown that elastase is also involved in G-CSF induced mobilization of stem cells. In the present study we examined the role of elastase in motility and proliferation of leukemic AML cells. We demonstrate a correlation between abnormal high levels of secreted elastase in the peripheral blood plasma of AML patients and the number of leukemic blast cells in the circulation. Interestingly, we found that AML cells not only secrete elastase, but also express constitutively membrane-bound elastase on their cell surface. Confocal microscopy revealed uniform localization of elastase on the cell surface. Of note, incubation of some AML cells with the chemokine SDF-1 increased elastase cell surface expression whereas treatment with neutralizing anti CXCR4 Abs decreased it. Pretreatment of primary human AML cells and cell lines with various elastase inhibitors and neutralizing anti elastase Abs led to reduced spontaneous and SDF-1-induced transwell migration of the leukemic cells. In vivo homing of AML mononuclear cells and CD34-enriched progenitors cells to the BM and spleen of transplanted NOD/SCID/B2mnull mice was significantly impaired by elastase inhibition. Moreover, when leukemic AML cells were pretreated with elastase inhibitors, the formation of spontaneous and SDF-1 induced protrusions was prevented suggesting that elastase participates in leukemic cell motility through direct regulation of cytoskeletal rearrangements and cell polarization. In addition, we found that the proliferation rate of AML cells was likewise elastase-dependent. In contrast, treatment of enriched human cord blood CD34+ progeniotrs with elastase inhibitors enhanced their proliferation rate, the percentage of undifferentiated CD34+/38− cells and their in vivo homing to the BM and spleen of immune deficient mice. Finally, we examined whether elastase may play a role in the abnormal AML cell egress from the BM to the circulation. Primary AML cells from different patients with different FAB subtypes were injected into sublethally irradiated NOD/SCID mice to establish human AML chimeras and 3–4 weeks later elastase inhibitor (1mg) was administrated daily for 4 consecutive days. Egress of AML cells to the blood circulation including blast cells was significantly decreased as compared to untreated chimeric mice. Taken together, these results demonstrate that elastase participates in the regulation of human AML progenitor cell proliferation and cell emigration from the BM to the circulation and that elastase inhibition can efficiently prevent AML cell egress in NOD/SCID chimeric mice.