ALBERT

Description: Azacitidine (AZA) is the standard of care for higher-risk myelodysplastic syndromes (MDS). Cytopenias are a major problem during the first treatment cycles due to MDS itself and to the mechanism of action of the drug, which includes not only demethylation, but also cytotoxicity and apoptosis. We evaluated the incidence of infectious complication during AZA treatment in 200 “real life” patients, collected retrospectively by the italian cooperative groups Gruppo Romano MDS (GROM) and Basilicata MDS Registry. Patients gave informed consent and the study was approved by the Ethycal Committes of partecipating Centers. Patient started AZA between 3/2006 and 3/2014. Median age at treatment start was 71 years (range 33-93 yrs, 66 females, 134 males). There were 182 MDS (up to 20% BM-Blasts) and 18 AML (median BM-blasts 23%, range 21-60% BM-blasts). In MDS, IPSS stratification (n=178) was: low: 4%, Int-1: 19%, Int-2: 60%, high 17%, and according to R-IPSS (n=123 pts): low: 4%, intermediate: 20%, high: 51%, very-high: 20%. MDS-specific comorbidity index (MDS-CI) was available for 154 patients: 50 patients were classified as low-risk (0), 85 as intermediate (1-2), and 19 as high risk (〉2). Azacitidine was started at a median of 1.4 months from initial diagnosis. One-hundred-sixty-six patients received AZA at the standard dose of 75 mg/sqm (7 days continuously: 22%, 5-2-2 days: 64%, 6-1-1: 4%) while 34 patients received AZA at 50 mg/sqm for 5- 7 days. Response, according to IWG 2006 criteria, was evaluable in 188 patients: 27 obtained complete remission (CR, 14%), marrow CR was achieved in 6 (3%), partial remission (PR) in 30 (16%), hematological improvement (HI) in 40 (21%), disease was reported stable (SD) in 54 (29%), whereas 31 patients presented progressive disease (16%). A median of 10 (range 1-62) cycles of AZA were delivered. Probability of response was independent from age, IPSS, IPSS-R, MDS-CI, and was associated to a shorter time from diagnosis to therapy initiation (p=0.04) and to the 75 mg/sqm through 7 days vs 50 mg/sqm through 5 -7 days schedule (60 vs 30% CR/PR/HI, p=0.007). With a median follow-up of 14.8 months (range 0-100 months), 57 patients are alive, resulting into a median overall survival of 17.2 months. IPSS and MDS-CI did not predict survival, while grouping according to IPSS-R was associated to a significantly different survival probability (p=0.0007). Across delivery of a total of 1547 AZA cycles, 213 (13%) febrile events were recorded. Fever of unknown origin was diagnosed in 20 patients (10%). Of 193 clinically documented events, a positive microbiologic test was available in 49 cases (25%, 44 for bacteria, 5 for viruses). A single episode of infection was recorded in 124 patients (62%) at a median of 3.8 months from therapy initiation (0-53 months), 53 (26.5%) patients suffered from a second infectious event and 28 (14%) patients from a third one (at 5.6 and 8.3 months from AZA start, respectively). Infections were the attributable cause of death in 30 patients (15%, 3 in CR, 17 with progressive disease and 10 SD). Most frequent infection sites were lungs (45%), cutis (9%), mouth (9%), bowel or peri-anal region (7%). The risk of infectious complications was lower in IPSS low-risk (p=0.05) or IPSS-R low/int MDS (p=0.01), and in patients with MDS-CI 0-2 (p=0.0001). Our data indicate that treatment with AZA is associated with a relatively high probability to develop infectious complications, especially pneumonia, which is however rarely the cause of death. The risk to develop infections is the highest during the first courses of AZA delivery and correlates to baseline patients’ characteristics, including disease stage and comorbidities. Once the infectious episode has occurred, the outcome depends on the disease status. Disclosures Voso: Celgene: Consultancy. Venditti:celgene: Consultancy. Breccia:novartis: Consultancy; BMS: Consultancy; Celgene: Consultancy.

Description: Abstract 3228 Although they frequently achieve complete remission (CR), adult patients with acute lymphoblastic leukemia (ALL) subsequently experience leukemia relapse, which represents an unresolved therapeutic problem. Based on the observation that ALL cells are frequently characterized by the deregulation of the apoptotic machinery, we and others have evaluated pre-clinically the activity of ABT-737 (kindly provided by Abbott Laboratories), a BH3-mimetic Bcl-2/Bcl-XL inhibitor, displaying a potent growth-inhibitory activity in ALL cell lines and primary cells. However ABT-737 binds to the anti-apoptotic protein Mcl-1 with low affinity and Mcl-1 expression may mediate resistance to ABT-737. Since ALL is also characterized by the aberrant activation of the mTOR and related signalling pathways, in the present study we further evaluated the combined Bcl-2/Bcl-XL (by ABT-737) and mTOR (by CCI-779) inhibition focusing, in particular, on the activity of combined molecularly targeted therapies on resistant cells. In MOLT-4 cells, ABT-737 induced dose and time-dependent growth inhibition (IC-50= 198nM) followed, at higher concentrations (250-500nM), by apoptosis induction. In contrast, the CEM-S, CEM-R, JURKAT, DAUDI and RAJI cells proved resistant (IC-50 〉5 μM). When we explored the effects of CCI-779 on the aforementioned cell lines, only minor cytostatic effects were observed (IC-50 0.5–28.2μM). MOLT-4 cells, for example, showed a flat dose-response curve (35-55% growth inhibition) at concentrations ranging between 1 and 5000 nM (IC50=9,87μM) and apoptosis induction was not seen until 5000 nM. We next investigated the effects of the combined use of ABT-737 and CCI-779 (each at 1000nM) in the ABT-resistant JURKAT cells. A significant (p= 0.04) induction of apoptosis was observed with the combination, as compared with single agents, after 24 h (47.7% ±5.9 of cells with sub-G1 DNA content with ABT-737 + CCI-779, compared to 17.4% ±1.5 and 4.2% ±1.5 with ABT-737 and CCI-779 as single agents, respectively). Similarly, when we exposed CEM-R cells to the drug combination (ABT-737 1000nM and CCI-779 5000nM) for 24 h, a strikingly stronger apoptosis induction (sub-G1 peak= 75.3% ±16.8) was observed, compared to single agents (15.8% ±7.2 and 4.2% ±1.9 with ABT-737 and CCI-779 alone, respectively) (p=0.0003). These effects were confirmed by measuring Annexin V binding. WB analysis showed decreased Mcl-1 levels, following exposure to CCI-779 and further downregulation in response to combined ABT-737+CCI-779 in the CEM-R cell line. These effects, however, were not seen in the parental CEM-S cell line. Primary cells, obtained from 10 ALL patients, showed an increase of the sub-G1 peak in 7/10 and in 4/10 samples, after exposure to ABT-737 (50nM) and CCI-779 (5000 nM), while synergistic effects on apoptosis induction were observed in 4/10 samples after exposure to the combination. In summary, we observed that the combined use of Bcl-2/Bcl-XL and mTOR inhibitors may exert synergistic cytotoxic effects in some resistant ALL models and this effect is associated with CCI-779-induced Mcl-1 down-regulation. Synergistic effects between these inhibitors were also found in a proportion of primary ALL samples, thus supporting further studies of combined Bcl-2/Bcl-XL and mTOR inhibitors, to overcome ALL resistance. Disclosures: Petrucci: Celgene: Honoraria; Janssen Cilag: Honoraria. Tafuri:Sigma-Tau: Research Funding.

Description: The Raf/MEK/ERK signaling module plays a pivotal role in the regulation of cell proliferation, survival, and differentiation. Our group, among others, has recently demonstrated that this pathway is frequently dysregulated in hematological malignancies and may constitute an attractive therapeutic target, particularly in AML. Here we investigated the effects of PD0325901, a novel MEK inhibitor, on phospho-protein expression, gene expression profiles, cell proliferation, and apoptosis in cell line models of AML, ALL, multiple myeloma (MM), ex vivo-cultured primary AML blasts, and oncogene-transformed hematopoietic cells. AML cell lines (OCI-AML2, OCI-AML3, HL-60) were strikingly sensitive to PD0325901 (IC50: 5–19 nM), NB4 (APL) and U266 (MM) showed intermediate sensitivity (IC50: 822 and 724 nM), while all the lymphoid cell lines tested and the myeloid cell lines U937 and KG1 were resistant (IC50 〉 1000 nM). Cell growth inhibition was due to inhibition of cell cycle progression and induction of apoptosis. A statistically significant reduction in the proportion of S-phase cells (p=0.01) and increase in the percentage of apoptotic cells (p=0.019) was also observed in 18 primary AML samples in response to 100 nM PD0325901. Analysis of the correlation between sensitivity/resistance to PD0325901 and Ras/Raf mutation status is currently ongoing. PD0325901 effects were also examined in a panel of IL-3-dependent murine myeloid FDC-P1 cell lines transformed to grow in response to 11 different oncogenes in the absence of IL-3. Fms-, Ras-, Raf-1-, B-Raf-, MEK1-, IGF-1R-, and STAT5a-transformed FDC-P1 cells were very sensitive to PD0325901 (IC50: ~ 1 nM), while A-Raf-, BCR-ABL-, EGFR- or Src-transformed cells were 10 to 100 fold less sensitive (IC50: 10 to 100 nM); the parental, IL-3 dependent FDC-P1 cell line had an IC50 〉 1000 nM. Analysis of the phosphorylation levels of 18 different target proteins after treatment with 10 nM PD0325901 showed a 5- to 8-fold reduction in ERK-1/2, observed only in sensitive cell lines, and a 2-fold reduction in JNK and STAT3 phosphorylation. PD0325901 (10 nM) treatment also profoundly altered the gene expression profile of the sensitive cell line OCI-AML3: 96 genes were modulated after 24 h (37 up- and 59 down-regulated), most of which involved in cell cycle regulation. Changes in cyclin D1 and D3, cyclin E, and cdc 25A were also validated at the protein level. Overall, PD0325901 shows potent growth-inhibitory and pro-apoptotic activity, indicating that MEK may be an appropriate therapeutic target in an array of different hematological malignancies. Further preclinical/clinical development of this compound is warranted, particularly in myeloid leukemias.

Description: Signaling pathways activated in erythroid cells have been recently characterized at the proteomic level by Reverse Phase Protein Array (RPPA) (Hricik T. et al., Am. J. Hematol. 2013). These experiments were performed on erythroblasts (Ery) obtained in HEMA (Human Erythroid Massive Amplification) cultures of healthy donors (adult blood, AB), CB and JAK2V617F positive PV patients. They showed that AB Ery express the highest levels of CD63, a member of the Transmembrane 4 Superfamily Proteins that quenches cKIT tyrosine kinase function and of cKIT phosphorylation at Y721 that mediates the interaction between cKIT and CD63 (Anzie et al, Blood 2002; 99: 4413). Since experiments in mice demonstrated that fetal cells are more responsive to cKIT stimulation by Stem Cell Factor (SCF) than the adult ones, we hypothesized that the stringency of the interaction between CD63 and cKIT may determine different levels of signaling activation and of proliferation in response to SCF in Ery from AB, CB and PV. To test this hypothesis, response to SCF of Ery obtained from an extended number of AB, CB and PV (〉5 subjects each) was characterized through biological (proliferation assays), biochemical (receptor downmodulation by FACS and confocal analyses) and signaling (RPPA) studies. Proliferation assays CB Ery achieved maximal proliferation with 30 ng/mL of SCF while maximal expansion of both AB and PV Ery required as much as 100 ng/mL of SCF. Anti-cKIT antibodies neutralized 100% of the proliferation response induced by 30 ng/mL of SCF in Ery from CB and AB but only 50% of that elicited by the same SCF concentration in Ery from PV. Receptor expression By FACS, SCF exposure downmodulated readily cKIT expression on Ery from CB (100% downmodulation with 10 ng/mL of SCF at 2 h), to a limited extend that on Ery from AB (100% downmodulation with 100 ng/mL at 2 h) and poorly that on Ery from PV (50% downmodulation with 100 ng/mL at 2 h). Confocal microscopy analysis showed that CD63 and cKIT were expressed in different proportions in Ery from AB, CB and PV with Ery from AB expressing the greatest CD63 levels and those from PV the greatest cKIT levels. Furthermore, upon SCF stimulation, CD63 and cKIT became strongly co-localized in Ery from AB but CD63/cKIT co-localization remained poor in Ery from CB and barely detectable in those from PV. Signaling studies RPPA determinations indicated that SCF stimulation induced different numbers of events in Ery from AB (8 hits), from CB (24 hits) and PV (42 hits). In AB Ery, SCF induced phosphorylation of cKITY721, of STAT3/STAT5 and of factors involved in transcription/translation (eIF4G, FKHR and S6 Ribosomal protein). In CB Ery, the numerous responses elicited by SCF stimulation included activation of mTOR/AKT pathway components (mTOR, AKT and p70 S6K). In PV Ery, SCF activated multiple signaling pathways involved in cell survival and expansion such as the mTOR pathway (mTOR, p70, S6 Ribosomal protein), the AKT pathway (FKHR, Glycogen Synthase Kinase 3 beta) and the Ras/MAPK pathway (c-Raf, ERK 1/2, Ras-GRF1, RSK3). We are currently validating by western blot and loss-of-function studies which of these pathways plays a major role in eliciting the abnormal amplification observed with Ery from PV. Conclusion These observations suggest that CD63 association with cKIT mediates the quenching of cKIT response occurring during the ontogenesis of human erythroid cells (CB-derived vs AB-derived Ery) and that this regulatory mechanism is disrupted as part of the pathogenetic process of PV. We propose that the signaling events identified by this proteomic profiling may represent targets to improve ex-vivo expansion of hematopoietic stem cells/progenitor cells or erythroblasts for cell therapy purposes or druggable targets for treatment of PV. Disclosures: No relevant conflicts of interest to declare.

Description: Epigenetic silencing of tumor suppressor (TS) genes is a hallmark in human leukemias, particularly through DNA methylation. Cyclin-dependent kinase inhibitors (CKI) are, among other genes, frequently found methylated in their promoter region. This epigenetic modification has been described also in acute lymphoblastic leukemia (ALL). However, the relationship between aberrant DNA methylation and protein expression of TS genes has not yet been extensively evaluated in adult ALL series. The aim of this study was to analyze in primary cells from newly diagnosed adult ALL patients, uniformly treated according to the LAL2000 GIMEMA protocol, the promoter methylation status of p73, p21, p15 and p16, evaluating in addition the p21, p15 and p16 protein expression. The DNA methylation status of promoter regions was investigated, according to cell availability, using a widely accepted method based on bisulfite modification of DNA, followed by methylation-specific PCR assay (MSP). Protein expression was evaluated by Western blot. Normal peripheral blood lymphocytes, as already described, resulted unmethylated for p73, p21, p15 and p16, and did not express the p21, p15 and p16 proteins. In ALL patients, in contrast, only the p21 promoter region was found constantly unmethylated. The p15, p16 and p73 promoter genes were found methylated in 15/37 (40.5%), 8/43 (18.6%) and 9/36 (25%) patients, respectively. Only 2/23 cases (8.6%) resulted simultaneously methylated for p15, p16 and p73. The p21 and p15 protein expression was found in 28/85 (32.9%) and 44/85 cases (51.8%), respectively. The p16 protein, in contrast, was never expressed. The p16 methylation was associated with the T-ALL (P=0.005) phenotype and with higher white blood cell (WBC) counts (P=0.027). Resistance to spontaneous induction of apoptosis was significantly associated with p21 protein expression (P=0.019) and its co-expression with p15 (P=0.049). Achievement of CR was not influenced by gene methylation status, nor by single protein expression. Interestingly, the co-expression of p15 and p21 was associated with failure to induction treatment: only 6/63 (9.5%) patients co-expressing p15 and p21 obtained a CR (P=0.027). Multivariate analysis confirmed the unfavorable role of this protein co-expression (P=0.059) on CR achievement. In contrast, once patients achieved remission, p21 protein expression was associated with a prolonged DFS, as confirmed by multivariate analysis for DFS (P=0.039). In conclusion, p15 and p21 protein expression plays an unfavorable prognostic role in adult ALL patients independently of the p73, p21, p15 and p16 gene promoter methylation status.

Description: The treatment of adult acute lymphoblastic leukemia (ALL) remains unsatisfactory. A potential hope is now given to Philadelphia-positive cases by targeted treatment modalities. Among other pathways involved in cell proliferation, we have recently demonstrated (Blood2007; 109:5473) the unfavorable role of ERK1/2 phosphorylation as an independent predictor of complete remission (CR) in adult ALL, suggesting the potential therapeutic value of other targeted therapies. The B-cell leukemia/lymphoma 2 (Bcl-2) family of proteins are important regulators of apoptosis and are frequently found aberrantly expressed, particularly in lymphoid malignancies. The role of Bcl-2 overexpression in tumorigenesis and chemoresistance prompted us to investigate whether the inhibition of the antiapoptotic function may result also in ALL in an attractive therapeutic strategy. In this study, we thus investigated the cell cycle and apoptotic effects of ABT-737 (kindly provided by Abbott Laboratories), a Bcl-2 (BH3) inhibitor, on both lymphoid leukemia cell lines and primary adult and childhood ALL cells. The lymphoid leukemia cell lines CEM and MOLT-4 were exposed to increasing concentrations of ABT-737 (from 0.1 to 1 μM) up to 72 hours. A dose- and time-dependent cell growth arrest and induction of apoptosis was found. In fact, measuring the subG0/1 peak at 48 hours, the levels of apoptosis increased in the CEM cell line from 14.1% (DMSO) to 34.4%, 64.5%, 86.5% and 98.6% at ABT-737 concentrations of 0.1, 0.25, 0.5 and 1 μM, respectively. Similarly, 48 hours of exposure to ABT-737 increased in MOLT-4 the Annexin V-positive cells from 7.2% to 64.2%. The effects of ABT-737 were then examined on primary blasts from 9 ALL patients (6 adults and 3 children). Bone marrow aspirates with a blast infiltration 〉70% were obtained at diagnosis from patients broadly characterized for clinical and biological parameters, as well as therapeutic response. ALL cells were cultured in vitro with ABT-737 (at increasing concentrations from 0.01 to 1 μM) for 24 hours. A significant decrease in viability was observed at 0.01 μM (p=0.008) with a remarkable dose-dependent increase of apoptosis. In fact, Annexin V-positive cells increased from a mean baseline value of 16.8% ± 8.8 to 43.6% ± 22.8 (p=0.04), 66% ± 21.3 (p=0.0001), 70.3% ± 26.9 (p=0.04), 74.6% ± 18.9 (p=0.03) and 76.2% ± 11.8 (p

Description: We show the molecular and functional characterization of a novel population of lineage-negative CD34-negative (Lin−CD34−) hematopoietic stem cells from chronic myelogenous leukemia (CML) patients at diagnosis. Molecular caryotyping and quantitative analysis of BCR-ABL transcript demonstrated that about one third of CD34− cells are leukemic. CML Lin−CD34− cells showed kinetic quiescence and limited clonogenic capacity. However, stroma-dependent cultures induced CD34 expression on some cells, cell cycling, acquisition of clonogenic activity and increased expression of BCR-ABL transcript. Lin−CD34− cells showed hematopoietic cell engraftment rate in immunodeficient mice similar to Lin-CD34+ cells whereas endothelial cell engraftment was significantly higher. Gene expression profiling revealed the down-regulation of cell cycle arrest genes, genes involved in antigen presentation and processing, while the expression of genes related to tumor progression, such as angiogenic factors, was strongly up-regulated when compared to normal counterparts. Flow cytometry analysis confirmed the significant down-regulation of HLA class I and II molecules in CML Lin−CD34−cells. Imatinib mesilate did not reduce fusion transcript levels, BCR-ABL kinase activity and clonogenic efficiency of CML Lin− CD34− cells in vitro. Moreover, leukemic CD34− cells survived to BCR-ABL inhibitors in vivo. Thus, we identified a novel CD34− leukemic stem cell subset in CML with peculiar molecular and functional characteristics.

Description: Introduction Effective therapies for R/R AML remain limited. MEK or MDM2 inhibition can downregulate MCL1, overcoming resistance to BCL2 inhibition. Preclinical synergy was seen when combining BCL2 inhibitor Ven with MEK inhibitor cobimetinib (cobi) or MDM2 inhibitor idasa (Han et al. ASH 2016; Pan et al. Cancer Cell 2017), supporting clinical evaluation in AML. Preliminary data in a Phase Ib dose-escalation study (NCT02670044) evaluating Ven+cobi/idasa in R/R AML suggested both combinations were tolerable (Daver et al. ASH 2017). However, Ven+cobi was closed due to limited clinical activity. Here we present data for additional pts, longer follow-up and biomarker analyses for Ven+idasa. Methods This ongoing, open-label, multicenter study evaluates safety, tolerability and efficacy of Ven+idasa in R/R AML or secondary AML previously treated for an antecedent hematologic disease. Pts 〉60 yrs of age and ineligible for cytotoxic therapy/allogeneic stem cell transplant were enrolled. A 2-dimensional dose escalation was used to establish the maximum tolerated dose: pts received doses of Ven orally (PO) daily (400mg or 600mg) + idasa PO daily on Days 1-5 (150mg, 200mg, or 400mg) in 28-day cycles. Plasma samples were taken for PK analysis at Cycles 1 and 2 Days 1 and 5, and Cycle 4 Day 1. BCL2, BCLxL and MCL1 status and minimal residual disease (MRD) were assayed centrally at Covance Laboratories using multicolor flow cytometry. Mutation (mut) sequencing was performed by Foundation Medicine using FoundationOne Heme at screening and from last bone marrow collected on study. Results As of April 6 2018, 34 pts received Ven+idasa across all dose cohorts (Table 1). Median age: 74 (range 64-93) yrs; median prior therapies: 1 (range 1-4); ECOG performance status 2: 18%; refractory: 56%; secondary AML: 53%; adverse cytogenetics: 27%. Pre-therapy mut data were available for 32 pts; most common muts were RUNX1 14 (41%), ASXL1 11 (32%), SRSF2 11 (32%). Other significant pre-therapy muts: TP53 6 (18%), IDH2 7 (21%), IDH1 1 (3%), FLT3 4 (13%). The most common adverse events (AEs) were diarrhea (88%) and nausea (71%); the most common grade (Gr) ≥3 AEs were neutropenia (32%), febrile neutropenia (32%), thrombocytopenia (29%; Table 2). After 2 cases of Gr 3 diarrhea in the Ven 600mg cohorts, mandatory prophylaxis was implemented; no further cases of Gr ≥3 diarrhea were seen in the following 10 pts. Laboratory tumor lysis syndrome occurred in 3 pts (9%); none required treatment discontinuation. There was no apparent PK drug-drug interaction between Ven and idasa. PK was dose-proportional over the ranges tested for Ven and idasa. The recommended Phase II dose (RP2D) has not been identified yet. Across all dose cohorts, 30/34 pts were response-evaluable; the remaining 4 were still on study treatment without post-baseline response assessment. The anti-leukemic response rate (CR+CRp+CRi+MLFS+PR) was 37% (11/30). Across the 2 Ven 600mg cohorts, which are being considered for RP2D, the anti-leukemic response rate was 9/18 (50%) (Table 1, Figure 1). MRD negativity (

Description: The aberrant function of transcription factors and/or kinase-based signaling pathways that regulate the ability of hematopoietic cells to proliferate, differentiate, and escape apoptosis accounts for the leukemic transformation of myeloid progenitors. Here, we demonstrate that simultaneous retinoid receptor ligation and blockade of the MEK/ERK signaling module, using the small-molecule inhibitor CI-1040, result in a strikingly synergistic induction of apoptosis in both acute myeloid leukemia (AML) and acute promyelocytic leukemia (APL) cells with constitutive ERK activation. This proapoptotic synergism requires functional RAR and RXR retinoid receptors, as demonstrated using RAR- and RXR-selective ligands and RAR-defective cells. In the presence of MEK inhibitors, however, retinoid-induced chromatin remodeling, target-gene transcription, and granulocytic differentiation are strikingly inhibited and apoptosis induction becomes independent of death-inducing ligand/receptor pairs; this suggests that apoptosis induction by combined retinoids and MEK inhibitors is entirely distinct from the classical “postmaturation” apoptosis induced by retinoids alone. Finally, we identify disruption of Bcl-2–dependent mitochondrial homeostasis as a possible point of convergence for the proapoptotic synergism observed with retinoids and MEK inhibitors. Taken together, these results indicate that combined retinoid treatment and MEK blockade exert powerful antileukemic effects and could be developed into a novel therapeutic strategy for both AML and APL.