ALBERT

Description: Abstract 1920 The biologic mechanisms involved in the pathogenesis of multiple myeloma (MM)-induced osteolytic bone disease are poorly understood. Physiological interactions between the serotoninergic and skeletal systems have been implicated by clinical observations. Brainstem-derived serotonin positively regulates bone mass following binding to 5-HT2C receptors on ventromedial hypothalamic neurons. This is opposed by platelet-derived serotonin that induces bone lysis and osteoclast activation. Moreover, the serotonin transporter SLCA6A4 is universally present among the malignant B cell clones. We examined serotonin dysregulation in two sample types from MM patients: peripheral blood and bone marrow. In the blood we measured by ELISA the ratio of serotonin in serum compared to platelets in patients with MM (n=10), MGUS (n=10) or healthy controls (n=5). We found higher levels of serotonin in platelets for the MM patients compared to the MGUS & healthy controls (p=0.017). Concomitantly there was less serotonin in the serum of MM patients compared to compared to the MGUS & healthy (p=0.002). This implies an imbalance in the compartmentalization of serotonin associated with presence of MM bone disease. Our multiplexed protein kinase signal pathway mapping technology, reverse phase protein microarrays (RPMA), was applied to bone marrow samples for quantifying post-translational modifications (e.g. phosphorylation, cleavage, acetylation) and/or total cell signaling kinase levels. Using reverse-phase protein microarray (RPMA) we retrospectively measured bone remodeling signal pathway perturbations in 15 bone marrow core biopsies from patients diagnosed with MM, at different clinical stages, and correlated this with the presence of MM-related bone disease (documented by scan or MRI). Bone marrow core biopsies exhibited significant elevation of cellular Serotonin, RANK, MMP-11, TNFα, TNF-R1, and Ezrin Tyr353 in MM with active bone disease (n=9) compared to patients without bone disease (n=6) (respectively p=0.031, p=0.038, p=0.0082, p=0.0221, p=0.01, p=0.028). To further evaluate the serotonin dysregulation in bone marrow cells, we measured serotonin bound to plasma cells (CD138+) compared to CD138- cells in bone marrow aspirates (n=21). Bone marrow aspirate were collected from patients undergoing standard of care hematological work up for multiple myeloma at any stage or treatment course. Patients with symptomatic myeloma (defined as presence of at least one of CRAB symptom) had lower serotonin levels in the CD138- bone microenvironment cells compared to non-symptomatic patients (p=0.0235). Plasma cells (CD138+) exhibited larger amounts of serotonin compared CD138- bone microenvironment cells (p=0.016). Taken together, our data show a dysregulation of serotonin in MM suggesting an altered distribution of serotonin in blood and bone marrow. This provides potential insights into diagnosis, prognosis, and/or treatment. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2860 Poster Board II-836 Multiple Myeloma (MM) is a heterogeneous disease, and despite significant advances in therapy it remains incurable. A major challenge facing clinicians is to determine which new agent or which combinations of agents will prove efficacious for individual patients. Interaction of MM cells with bone marrow microenvironment cells has a pathogenetic role in the disease and confers tumor cell resistance to conventional therapies. The new generation of therapies is designed to target signal transduction pathways, particularly tyrosine kinase signaling. The phosphorylation, or activation state, of kinase-driven signal networks contains important information concerning disease pathogenesis and the ongoing state of kinase-associated therapeutic targets. Reverse-phase protein microarray (RPMA) is a reproducible, high-throughput system for protein signal pathway profiling. Phosphorylation state of kinase-associated therapeutic targets provides direct information regarding the target and off-target effects of treatment. We describe herein an ex vivo study of signal pathway inhibitor treatments of fresh bone marrow aspirate samples from patients undergoing standard of care hematological work up for multiple myeloma at any stage or treatment course. Bone marrow aspirates (n=37 to date), not required for diagnosis, were immediately subdivided and treated ex vivo with a panel of molecular targeted inhibitors and combinations, and/or exogenous ligands (SCF, IGF-1), and/or cytokines (IL-6), that target a wide range of cellular pathways (e.g. proteosome, angiogenesis related, protein degradation, cell proliferation/survival, insulin response, and protein translation). Up to 48 different treatment conditions can be studied for each patient sample from a bone marrow aspirate volume of 5.0mL. The objectives were a) measure the signal pathway perturbations caused by the inhibitor/ligand treatment in individual bone marrow aspirate samples, b) compare the relative sensitivity of tumor and non tumor bone marrow cells treated in admixture under identical conditions to identify predictive/prognostic protein-based biomarkers. We specifically measured the post-translational modifications (i.e. phosphorylation) of the drug target substrates to provide direct information regarding the target and off-target effects of treatment. After incubation for 4 hours the bone marrow aspirate samples were placed in a preservative that suppresses fluctuations in kinase pathway proteins. MM CD138 positive cells were separated from the non-CD138+ bone marrow microenvironment cells via immunomagnetic sorting. RPMA was used to quantitatively map 75 cell signaling pathway endpoints in CD138+ and non-CD138+ cell populations that were treated simultaneously prior to cell sorting. To date the accrual has included untreated patients with MGUS, smoldering myeloma, treatment naïve, or advanced stage, pre-treated myeloma. Individual patients differed widely in response to ex vivo treatment and combination of drug treatments. Dexamethasone showed differential effects on the myeloma cells compared to non-myeloma cells from the same patient. For all the untreated patients studied to date, dexamethasone significantly suppressed the phosphoAKT or NF-KB endpoints, while increasing the pro-apoptotic signaling proteins such as cleaved caspases. In contrast, all the patients heavily pre-treated with dexamethasone in combination with other agents did not show any differential effects of the dexamethasone treatment on the CD138+ cells compared to the non-CD138+ cells in the bone marrow microenvironment. Many molecular targeted inhibitors induced an up-regulation of the target pathway even in combination therapies such as Rapamycin plus an IGF-1R inhibitor. Sorafinib, a molecular targeted tyrosine kinase inhibitor which blocks the MAPK (Raf/Mek/ERK) pathway, induced a compensatory up-regulation of ERK T202/Y204 in all patients except one who was in remission after treatment. Multiplexed phosphoprotein cell signaling analysis before or after treatment with ex vivo inhibitors may predict patient-specific therapeutic response and/or off target effects. Selected agents that show efficacy in ex vivo studies may be considered for further in vivo clinical studies in which signal profiling of the bone marrow cellular populations could be performed before and after molecular targeted therapy. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4468 Suppression of BCR-ABL1 catalytic activity by the tyrosine kinase inhibitor (TKI) Imatinib Mesylate (IM) has dramatically improved the natural history of Chronic Myeloid Leukemia (CML) and - to date - represents the first and most successful example of effective anti-cancer targeted therapy {Druker, 2009}. Despite the success, patients can become resistant. Since IM-resistance in CML patients occurs despite adequate suppression of BCR-ABL activity, it is likely due to the activation of other pathways, and for this reason we need to discover novel Bcr-Abl independent pathways than can become the targets of resistant cells. Therefore we used immortalized CML human cell lines K562 and LAM84, both sensitive and resistant to Imatinib, to study the signaling in sensitive/resistant cell lines following treatment with 4 different compounds. Reverse-Phase protein microarrays were used to quantitatively map 35 cell signaling pathway endpoints, including survival, proliferation, drug resistance, apoptosis, and autophagy. Cells were incubated with IM 1uM, Dasatinib 1uM, Nilotinib 1uM or LY-29400210 uM and after 2 or 12 hours were placed in a preservative that suppresses fluctuations in kinase pathway proteins (Espina, 2008). 5/35 protein endpoints were linked together and suppressed by Dasatinib, even in the resistant cell line: PLC-y-1-Tyr783, and its upstream (ShCTyr317 SrcTyr416) and downstream targets (mTORSer2448, STAT5Tyr694, ERKThr202/Tyr204) without interfering with AKT activation status on Ser473 compared to Imatinib (p=0.0031 for K562, p= 0.042 for LAM84), Nilotinib (p=0.0034 for K562, p= 0.043 for LAM84), LY-294002 (p=0.0009 for K562, p= 0.015 for LAM84). In Imatinib-sensitive cell line LAM84 there were no differences between Dasatinib and Imatinib in the modulation of the pathway, compared to IM-sensitive K562 cell line (p=0.005), thus confirming the different profile among these two CML models. Dasatinib showed a greater suppression of the PLC-y-1 pathway compared to LY-294002 in both resistant cell lines (K562, p=0.0009 and LAM84, p=0.015). These data confirm and extend the conclusions of Markova et al. (Oncogene 2010) showing PLC-y-1 as a mechanism of death in sensitive cells. Our data showed the mechanism by which second generation TKIs (dasatinib) can overcome Imatinib resistance by suppressing the PLC-y-1 pathway. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3818 Background Conventional cytogenetics is the major prognostic factor in predicting outcome of myelodysplasia (MDS), but at least 50% of patients have a normal karyotype. 5-azacytidine (5-AZA) is a DNA methyltransferase inhibitor used in treatment of high-risk MDS, which is able to delay the progression to AML and improve clinical outcomes, but targets of the methylation status are poor known. Aims To evaluate molecular changes, at genomic or proteomic level, which can be identified as additional targets of 5-azacytidine in MDS patients with normal conventional cytogenetics. Methods By reverse-phase protein microarray (RPMA), we analyzed bone marrow mononuclear cells from 19 patients affected of high risk MDS, treated with 5-azacytidine (median age 71 years, M/F=12/5). Treatment consisted of 4 cycles of 100mg flat dose for 7 days+21 days of wash-out. In 7 cases the sample after 4 cycles of therapy was matched to the sample collected before starting treatment. RPMA was used to quantitatively map 45 cell signaling pathway endpoints, including survival, proliferation, drug resistance, apoptosis, and autophagy. For the first 4 patients we used also a genome-wide approach based on SNP (single nucleotide polymorphism) array (6.0 Affymetrix platform) to detect copy number and allelotype data in order to identify new genetic markers in terms of SNP, CNV and LOH. Results All patients were evaluable for response one month after the 4th cycle. Three patients were refractory and progressed to AML and 1 was a late responder (after 7 cycles). All other patients experienced hematologic improvement. After 4 cycles of 5-AZA, at proteomic level we found three main signaling pathways were increased and did not correlate with the response: 1) pro-survival signaling: PLC-y-1-Tyr783 (p=0.0017), and its upstream regulators Src-Tyr416, c-Abl-Tyr735 (p=0.002) and downstream target STAT5Tyr694 (p=0.0017) were increased, without affecting proliferative pathways, such as AKT activation status on Ser473 and Thr308 or mTORSer2448. 2) Autophagy: ATG5, Beclin 1 and LC3B were significantly elevated after treatment (p values respectively

Description: The outcome for children with Acute Lymphoblastic Leukemia (ALL) has improved in the last few decades with current therapy resulting in an event free survival exceeding 75%. Despite of this, 25% of patients still relapse or resist to conventional therapies. Proteomics, in particular the Reverse Phase Protein Arrays (RPA), offers a new approach to study both the potential molecular mechanisms of drugs chemosensitivity versus resistance, and identify new drug targets in the patients that do not respond to therapy. RPA, which can quantitatively measure dozens to hundreds of phosphorylated kinase substrates from only a few thousand cells, can be used to profile the working state of cellular signaling pathways in a manner not possible with gene microarrays, since post-translational modifications cannot be accurately portrayed by global gene expression patterns alone. We employed RPA to study the phosphorylated cellular pathways in bone marrow aspirate samples collected prior to treatment. The phosphorylation status of 95 key signaling proteins, including pro-survival, pro-apoptotic and those involved in cell cycle control were analyzed from 120 pediatric B-ALL specimens collected at the Pediatric Oncohematology Laboratory (University of Padova) over the past 15 years. Molecular network analysis and phosphoprotein profiling were performed using commercially available software. Our results indicate that there may be a difference between the molecular networks of activated kinase substrates within leukemic patients depending on disease state, genotype, or response therapy. The discovery of the molecular mechanisms related to drug resistance could play an important role for individualizing therapy and may reveal new strategies to improve treatment outcome.

Description: Abstract 930 Chronic lymphocytic leukemia (CLL) cell growth and survival occur in defined microenvironment niches controlled by several receptor/ligand interactions including those mediated by the α4β1 integrin. This integrin, in particular, is known to interact with both the extracellular matrix component fibronectin, via the non-RGD CS-1 fragments, and the bone marrow/stromal components, via the vascular-cell adhesion molecule-1 (VCAM-1). The α4 integrin chain (a.k.a. CD49d) has been previously shown to be associated with poor prognosis in CLL by marking a subset of CLL patients characterized by a more aggressive clinical course both in term of disease progression and overall survival. Despite the great deal of studies investigating CLL cell microenvironmental interactions in tissue sites, little is known regarding the constitutive engagement of adhesion receptors in circulating CLL cells, and the role in this context of plasma/plasma components. To address this issue, the proteomic profiles of circulating leukemic cells from 80 CLL cases expressing or not CD49d were explored using a reverse phase protein microarray (RPMA) approach quantitatively analysing 77 proteins (61 phosphoproteins) known to be involved in translational control, cell growth, apoptosis, B cell receptor and cytoskeletal signaling. Comparison of the signaling activation portrait between 40 CD49d pos and 40 CD49d neg CLL cases highlighted the over-expression in CD49d pos CLL of proteins involved in the regulation of integrin-mediated cytoskeletal dynamics, such as phospho-p21-activated kinase (PAK1 Ser199-204/PAK2 Ser192-197; p=0.0005), phospho-LIM kinase (LIMK1 Thr508/LIMK2 Thr505; p=0.00001) and the adaptor protein CrkII Tyr221 (p=0.039). Moreover, CD49d pos CLL cells were characterized by a high correlation between proteins involved in integrin-mediated signal trasduction, including the focal-adhesion kinase (FAK Tyr576-Ser577), the tyrosine kinase Src Tyr527 and the adaptor protein CrkL Tyr207. Since PAK and LIMK represent key players in the modulation of the structure and the activity of actin cytoskeleton, we focused on these proteins for validation experiments. The constitutive pPAK and pLIMK overexpression in the CD49d pos CLL group was confirmed by western blot analysis comparing purified CLL cells from 3 CD49d pos versus 3 CD49d neg cases (mean fold increase 〉100 for both proteins). The results obtained suggest that integrin signalling is constitutively active in CD49d-expressing circulating CLL cells, pointing to a constitutive receptor engagement by CD49d ligands allegedly present in plasma. To test whether plasma constituents could modulate integrin-signaling proteins, CLL cells from 5 cases, expressing or not CD49d, were challenged with autologous plasma (1:3 dilution) for 30 seconds, 1 and 3 minutes. The presence of plasma induced a strong increase of PAK and LIMK phosphorylation intensities in CD49d pos CLL cells, starting at 30 seconds upon stimulation (mean fold increase 〉10 as compared to control for both proteins), and increasing after 1 and 3 minutes (mean fold increase 〉40 and 〉50 for both the proteins, respectively). Conversely, plasma stimulation did not induce pPAK and pLIMK expression modulation in CD49d neg CLL cells. Of note, pretreatment of CD49d-expressing CLL cells with the anti-CD49d HP1/2 blocking antibody, resulted in lower up-regulation of pPAK and pLIMK with an overall 60% inhibition for both the proteins (p=0.01), confirming the involvement of CD49d triggering in the observed activated signaling. Given the above results, we investigated plasma from 24 CLL patients, expressing CD49d at different levels, for the presence of the CD49d ligand fibronectin using an ELISA detection kit. All samples tested showed more than 1 mg/ml fibronectin concentration, without differences between CD49d-expressing and CD49d neg CLL cases. Altogether these results sustain the hypothesis of an active role of plasma/plasma components in the activation of CD49d-mediated integrin pathway, thus favoring the delivery of chemoresistance/pro-survival signals even in the context of circulating CLL cells. Our results may be of interest in the perspective of novel therapies (e.g. Bruton tyrosine kinase inhibitors) known to provoke a massive egress of neoplastic cells from tissue sites into the blood stream. Disclosures: No relevant conflicts of interest to declare.