ALBERT

Description: Introduction: Multiple myeloma is a heterogeneous plasma cell neoplasm that remains all but incurable despite significant advances in treatment. We anticipate that the ability to overcome this hurdle resides in personalized strategies designed to specifically recognize, target, and anticipate dynamic tumor subpopulations with variable drug response profiles within an individual. To this end, we have developed a novel multi-disciplinary approach using organotypic drug screening and mathematical modeling to assess drug sensitivity of the different subpopulations within the tumor burden of individual patients and, in turn, provide accurate predictions of clinical outcome to anti-myeloma therapy. Material and methods: We have used a novel combination of ex vivo drug sensitivity assay and mathematical models to predict clinical response of 48 MM patients (11 newly diagnosed and 37 relapsed, 18 females and 30 males, median age 64.5, range 45-77) treated with a combination of proteasome inhibitors and IMIDs (37), nuclear export and topo2 isomerase inhibitors (10), and high dose melphalan (1). MM cells (CD138+) were extracted from fresh bone marrow aspirates and seeded in an ex vivo co-culture model with human stroma in 384-well plates. These cells were exposed to a number of chemotherapeutic and experimental agents (up to 31) for a period of 4 days, during which viability was assessed continuously using bright field imaging and digital image analysis. A mathematical model was used to interpolate the dose response dynamics to each drug, and combined with drug and regimen-specific pharmacokinetic data, generate predictions of clinical response to each individual drug. We have then validated ex vivo-based predictions with actual outcome 90 days post-biopsy. In patients treated with combinations, the mathematical model combined the effect of each single drug assuming additivity. Results: To examine the accuracy of the predicted in silico responses, we have assessed the model according to three increasingly strict standards of accuracy: (A) The model correctly predicted 32 out of 32 responders (100%) and 14 out of 16 non-responders (88%), with an overall accuracy of 96%; (B) According to IMWG stratification, the model correctly stratified 14 out of 16 patients as stable or progressive disease (PD/SD, 88%, the remaining 2 incorrectly predicted as MR/PR), 15 our of 18 as minimal or partial response (MR/PR, 83%, the remaining 3 incorrectly predicted as VGPR/CR), and 10 out of 14 patients as very good partial response or complete response (71%, the remaining 4 incorrectly classified as MR/PR), with an overall accuracy of 81%; (C) The 48 patients from this study provided a total of 120 measures of tumor burden (M-spike or SFLC) within the 90-day post-biopsy period. The direct correlation between tumor burden measures and model predictions led to a Pearson r=0.5547 (P

Description: Multiple Myeloma (MM) is an incurable disease that warrants novel treatments. MM survival is dependent on resistance conferred by interactions with the bone marrow (BM) microenvironment (stroma and/or extracellular matrix proteins): Environment Mediated-Drug Resistance (EM-DR). We have shown that adhesion to fibronectin inhibits CD95-induced caspase-8 activation resulting in apoptosis by regulating the cellular localization and availability of c- FLIPL (Shain K. et al. J Immunol. 2002;168(5):2544–53). TNF-related apoptosis inducing ligand/Apo-2L (TRAIL/Apo-2L) is a member of the TNF superfamily of death ligands that induce apoptosis of resistant MM cell lines and patient’s cells while cultured in suspension. TRAIL/Apo-2L also inhibits the growth of human plasmocytomas in xenografted NOD/SCID mice (Mitsiades C. et al. Blood2001; 98(3):795–804). To explore if the tumor microenvironment confers resistance to TRAIL/Apo-2L mediated apoptosis, we treated RPMI 8226 cells with recombinant human killer TRAIL (Alexis Biochemicals). In a series of 3 experiments, we observed a 50.8 ± 10 % specific apoptosis, measured by annexin/PI staining, when cells were treated with TRAIL (10ng/mL) in suspension (Su) media. In contrast, a dramatic resistance to TRAIL mediated apoptosis (6.9 ± 1.2 %) was observed when RPMI 8226 cells were adhered (Ad) to stroma cells (HS-5 and HS-5 transfected with green fluorescent protein-GFP) suggesting the EM-DR phenotype. We hypothesized that EM-DR can potentially be reversed by Bortezomib, a proteosome inhibitor, via NF-κB inhibition resulting in reduced transcription of cellular adhesion proteins genes (Mitsiades et al. Blood2002; 99(11): 4079–4086). Resistance to drug-induced apoptosis was also observed when RPMI 8226 cells were adhered to HS-5 and HS5-GFP and treated with Bortezomib alone at 5, 10 and 15nM (55.6 ± 11.6 % Su and 12.1 ± 1.6 % Ad). Microarray analysis of RPMI 8226 cells treated with Bortezomib showed that death receptor 4 (DR4) and DR5 expression was up-regulated suggesting a potential interaction between Bortezomib and TRAIL pathway. When RPMI 8226 cells were treated with Bortezomib (10nM) for 20 Hrs and subsequently treated with TRAIL for additional 4 Hrs, we observed an enhancement to 77.7 ± 3.64 % (Su) and 47.22 ± 13.6 % (Ad) specific apoptosis. In addition, in a series of 3 experiments using transwell inserts, RPMI 8226 cells (upper well) separated from HS-5 (lower well) were treated with Bortezomib (10nM), TRAIL (10ng/ML) or Bortezomib and TRAIL combined as described above. Results were comparable with direct contact experiments described, suggesting that cytokines and/or chemokines released by stroma may be responsible for EM-DR phenotype (Figure 1). Further mechanistic studies elucidating TRAIL and Bortezomib combination are currently under investigation. These preclinical studies provide the basis for clinical trial testing the combination of these agents. Figure Figure

Description: SH2-containing-5′inositol phosphatase-1 (SHIP), via removal of the 5′ phosphate group from PI(3,4,5) P3 (PIP3), influences signals downstream of cytokine/chemokine receptors that play a key role in megakaryocytopoiesis. Thombopoietin (TPO) influences megakaryocytes (MK) development by controlling their proliferation, differentiation and survival. SHIP phosphorylation, resulting from TPO receptor activation (c-mpl) influences MK cycling and proliferation. Stromal derived factor-1 (SDF-1/CXCL12) induces transendothelial MK migration facilitating platelet shedding. SHIP-deficient myeloid progenitors exhibit enhanced chemotaxis towards SDF-1/CXCL-12, indicating SHIP influences signaling downstream of its receptor (CXCR-4). In addition, colony-forming-unit-MK are decreased in SHIP−/− bone marrow (BM) and SHIP regulates PIP3 levels after thrombin or collagen platelet activation. To further explore SHIP effects on megakaryocytopoiesis, we measured MK compartment size, by flow cytometry, in mice with SHIP promoter/first exon deletion (SHIP−/−) and with inositol phosphatase deletion (SHIPΔIP/ΔIP) in order to confirm that the observed phenotype is highly penetrant. Lin− cKit+ CD41+ (n=5/strain), representing MK progenitors (MKP), were statistically significantly increased in BM (3-fold in SHIP−/− and SHIPΔIP/ΔIP), spleen (18-fold in SHIP−/−; 50.8-fold in SHIPΔIP/ΔIP) and peripheral blood (PB) (2.4-fold in SHIPΔIP/ΔIP) compared to their wild type (WT) littermates; not reaching statistical significance in SHIP−/− PB (1.6-fold). These findings suggest that SHIP may control MKP homeostasis. Lin− cKit- CD41+ (n=5), representing mature MK, were statistically significantly decreased in BM (2.6-fold in SHIP−/−; 2.2-fold in SHIPΔIP/ΔIP), increased in spleen (11-fold in SHIP−/−; 26.7-fold in SHIPΔIP/ΔIP) and PB (7.7-fold in SHIP−/−; 2.6-fold in SHIPΔIP/ΔIP); suggesting that SHIP may control MK redistribution. BM histopathology and Glycoprotein IIB/IIIa (CD41) immuno-staining showed that MK numbers were similar in SHIP−/− and WT, although morphologically we detected hypolobulated/micro (HM) MK in SHIP−/− and hyperlobulated (HL) MK in WT mice, consistent with MKP and MK flow cytometry phenotype respectively. Spleen histopathology and CD41 immuno-staining showed that the MK numbers were increased in SHIP−/−; cells with HM and HL morphology were present. These findings suggest that SHIP may control pathways that mediate MK localization and/or migration. Mean platelet numbers (0.95 x 106/μL SHIP−/−; 0.75 x 106/μL SHIPΔIP/ΔIP) were not significantly different compared to WT (1x 106/μL SHIP−/− and SHIPΔIP/ΔIP) although splenomegaly in SHIP−/− animals may prevent an increase in circulating platelets. In conclusion, SHIP regulates essential signaling pathways that control megakaryocytopoiesis in vivo. SHIP enzymatic activity could be targeted to increase MKP pool to enable this compartment to replenish platelets more rapidly following chemotherapy and radiation treatment.

Description: Resistance to chemotherapy arises in a complex microenvironment and it is essential to study how it influences apoptosis in order to identify novel therapeutic targets. APO2L/TRAIL anti-tumor activity is attractive since it spares normal tissue. Previous studies in our laboratory have shown that cancer:stroma microenvironment interactions confer resistance to immune control via APO2L/TRAIL, a process referred to as environmental mediated-immune resistance (EM-IR). We studied EM-IR in multiple myeloma because it is a disease subject to bone marrow environmental influences and expresses the specific APO2L/TRAIL receptor(s). We have shown in a transwell assay, with myeloma (RPMI-8226, U266 and MM1s) in the upper well and HS5 stromal cells in the lower well, that APO2L/TRAIL (5 to 50ng/mL for 5 hours) apoptosis is reduced when compared to cells exposed to medium in the lower well. Additional studies using conditioned medium from HS5 and myeloma patient’s stroma (n=3) grown for 14 days, confirmed that soluble factor(s) produced by tumor bone marrow microenvironment protected cells from APO2/TRAIL apoptosis. APO2/TRAIL resistance was reversible as sensitivity was restored after HS5 stroma cells were removed (20 min). APO2/TRAIL signaling pathway studies showed that baseline levels of c-FLIP, an anti-apoptotic factor, increased in RPMI-8226 (2.86 fold), in U266 (9.5 fold increase) and in MM1s (1.34 fold increase) myeloma cell lines. Inhibition of c-FLIP by means of RNA interference using siRNA duplex that targeted both c-FLIP isoforms, c-FLIP long and short, increased APO2/TRAIL sensitivity of RPMI-8226 treated in the presence of HS5 stroma. Sub-cellular fractionation showed that c-FLIP is maintained in the membrane fraction and is not significantly increased in the cytosol when exposed to soluble factor(s). Pre-treatment (19 hours) with cyclohexamide, a protein synthesis inhibitor, restored APO2L/TRAIL sensitivity in association with down regulation of c-FLIP while maintained anti-apoptotic c-IAP, Bcl2 and Mcl-1 levels suggesting that c-FLIP synthesis, not intracellular traffic, is essential for soluble factors to regulate c-FLIP. To dissect which soluble factor(s) are involved in apoptosis resistance we have tested the effects of HS27a stroma cells. HS27a lack IL-6 and/or IL-1 production compared to HS5 stroma and do not confer APO2/TRAIL resistance suggesting that these cytokines may be involved in apoptosis resistance. Treatment with neutralizing IL-6 specific antibody of HS5 transwell decreased IL-6 levels and partially restored sensitivity to APO2L/TRAIL in myeloma. Treatment with rh-IL-6 (0.001ng/mL to 10ng/mL) with APO2L/TRAIL conferred a dose-dependent resistance to APO2L/TRAIL in myeloma cells associated with increased c-FLIP levels. IL-1 receptor antagonist (IL-1ra) either by pre-treatment (1hour–18 hours) or co-administration with APO2L/TRAIL did not reverse APO2L/TRAIL resistance. These findings suggest that IL-6 in part mediates APO2L/TRAIL EM-IR by increasing c-FLIP levels among other unknown mechanisms. We conclude that c-FLIP up-regulation inhibitors and/or targeting hematopoietic soluble factor(s) may contribute to enhanced APO2/TRAIL function.

Description: Abstract 3022FN2 Introduction of reduced toxicity regimens for hematopoietic cell allografting is expanding applicability of this procedure to patients not otherwise eligible to receive myeloablative preparative regimens. This phase II study evaluates the ability of a novel reduced toxicity regimen with pentostatin and busulfan (BU) to achieve ≥ 50% donor chimerism by day +28 (±7) in CD3+ blood lymphocytes. The preparative regimen consisted of a pre-conditioning treatment-phase with pentostatin 4 mg/m2 (days -28, -21, and -14) if host CD4+ counts 〉100/μL; and, rituximab 375 mg/m2 (days -21, -14, and -7) for CD20+ lymphoid malignancies. Conditioning phase consisted of pentostatin 4 mg/m2 (days -4, -3) and 2 doses of I.V BU: 1st dose (day -4) = 200 mg/m2, 2nd dose (day -2) = adjusted for a target cumulative area under the curve (AUC) =16, 000 micromol*min/L (±10%). Patients (pts) with CD20+ lymphoid malignancies received 2 additional rituximab doses of 375 mg/m2 (days +1, +8) post-transplant. Between 02/04/2008 and 03/18/2011, 42 pts [31(74%) male] with a median age of 53 (29–73) years, underwent allogeneic HCT for the following diagnoses: CLL/SLL=18 (43%), follicular NHL=6 (14%), mantle cell NHL=4 (10%), transformed B-cell NHL=3 (7%), DLBC NHL=2 (5%), PTCL NOS=2 (5%), HL=2 (5%), lymphoplasmacytic NHL=1 (2%), T-cell PLL=1 (2%), and AML/MDS=3 (7%). Disease status was CR1/2 (n = 4), CR3+ (n = 5), PR1/2 (n = 15), PR3+ (n = 8), and refractory disease (n = 10). Seventy six percent of cases had a Karnofsky performance score of ≥ 90. All pts received G-CSF mobilized PBSC from 20 MRD, 18 MUD, and 4 MMUD (7/8 antigen/allele). Median CD34+ dose infused was 8.93 (1.66 − 14.07) x106/kg cells. GVHD prophylaxis consisted of TAC-MTX in 36, TAC-sirolimus in 4, and TAC-MMF in 2 cases. No pts received ATG. Thirty-three pts (79%) received rituximab. Median (range) CD4 levels/μL on days -30, -23, -16, -7, and 0 were 342 (27–844), 165 (33–642), 116 (41–413), 129 (43–344), and 31 (9–161), respectively. BU AUC [median (range)] was 8204 (5830 – 12116) for dose 1, 9303 (4763 – 13433) for dose 2 and 17518 (15010 – 21215) for the total dosing interval. Neutrophil and platelet engraftment occurred at a median (range) of 17 days (15 – 28) days and 13 days (0 – 177), respectively. Donor CD3, CD33 and unsorted BM chimerisms [median (range)] on day 28 (±7) were 87% (61–100%), 100% (95–100%), and 96% (64–100%), respectively. The median (range) follow-up of survivors was 23.8 (3.4–38.6) months. Non-relapse mortality at 100-days and 2-years were 2.4% (95%CI: 0 –9.1%) and 18.6% (95%CI: 7.8–32.6%), respectively. Overall response rate was 86% (CR=74%, PR=12%). Two-year progression-free (PFS) and overall survivals (OS) for all pts were 56.4% (95%CI: 40.4–71.7) and 63.5% (95%CI: 46.7–78.8%), respectively. Disease status (CR, PR or refractory) at time of HCT did not affect 2-year PFS (p=0.83) or OS (p=0.97). Cumulative incidence of grade II-IV and III-IV GVHD by day +100 were 59% (95% CI: 43 – 75%) and 19% (95% CI: 7 – 35%), respectively. Cumulative incidence of moderate/severe chronic GVHD at 2 years was 58% (95% CI: 39 – 75%). In summary, our results demonstrate that this novel conditioning regimen of PB±R facilitates early durable CD3+ donor engraftment and results in excellent response rates with low NRM. Disclosures: Off Label Use: Pentostatin, Busulfan and Rituximab as conditioning regimens for allogeneic HCT. Perkins:PDL BioPharma: Research funding. Fernandez:Genetech: Advisory board; Otsuka: Speakers Bureau.

Description: TNF-related apoptosis inducing ligand (TRAIL), a member of the TNF superfamily of death ligands, is preferentially cytotoxic against neoplastic cells and suppresses tumor development in vivo. TRAIL plays a role in Natural Killer, TNF-activated T and Dendritic cell-dependent tumor surveillance. The bone marrow microenvironment provides tumor protection from chemotherapeutic agents and Fas-death receptor signaling, a process known as environmental mediated drug resistance (EM-DR). In this study we have investigated whether EM-DR confers resistance to apoptosis mediated by TRAIL in Multiple Myeloma. We hypothesize that EM-DR contributes to evade TRAIL mediated apoptosis resulting in Multiple Myeloma progression. Three drug sensitive Mulitple Myeloma cell lines (RPMI-8266, U266 and MM1s) exhibits a dramatic apoptosis resistance to recombinant human TRAIL (10ng/mL) after twenty four hours of exposure when directly attached to HS-5 stromal cells as determined by annexin/7-AAD staining (Table 1). RPMI-8226 TRAIL-mediated apoptosis was demonstrated dose- and time-dependent. Experiments in transwell assay with RPMI-8226 in the upper well and HS-5 in the lower well induced significantly reduced apoptosis (8.9 ± 5.4 %) compared to RPMI-8226 cells exposed to medium in lower well (37 ± 16 %) after four hour treatment with TRAIL (10ng/mL). Western blot analysis of RPMI-8226 cells exposed to HS-5 for four hours in transwell exhibited attenuated caspase-8, caspase-3 and PARP cleavage. Direct contact of RPMI-8226 cells with bone marrow fibronectin did not confer resistant to TRAIL. These findings suggest that cell:cell contact and soluble factors produced by stromal cells contribute to TRAIL EM-DR. Pretreatment of RPMI-8226 cells with proteosome inhibitor Bortezomib (10nM) partially overcomes TRAIL EM-DR in cells directly attached to HS-5 and in transwell assays with HS-5 in lower well (Table 2). Bortezomib up-regulated TRAIL receptor-2 (DR5) mRNA expression in RPMI-8226 cells, but did not increase DR5 protein expression as determined by Western blot and flow cytometry. We hypothesize that NF-κB inactivation by bortezomib reduces transcription of genes that mediate TRAIL EM-DR. Mechanistic studies elucidating TRAIL EM-DR are under investigation with the purpose to identify novel targets to enhance anti-tumor activity of TRAIL in vivo. Table 1. Adhesion of RPMI-8226, U266 and MM1s Myeloma Cells to HS-5 Stromal Cells Attenuates TRAIL Mediated Apoptosis RPMI-8226 (% Specific Apoptosis) U266 (% Specific Apoptosis) MM1s (% Specific Apoptosis) Suspension 75±10 % 11±2.7 % 25±1.7 % Direct Adhesion to HS-5 stromal cells 12±1.8 % 3.5±1 % 4±0.7 % Table 2. Bortezomib Partially Overcomes RPMI-8226 Environment Mediated-TRAIL Resistance Direct Adhesion to HS-5 stromal cells (% Specific Apoptosis) Transwell Assay with HS-5 in lower well (% Specific Apoptosis) *TRAIL (10ng/mL) for 4 hours ** Bortezomib (10nM) for 20 hours followed by TRAIL (10ng/mL) + Bortezomib for 4 hours TRAIL * 1.5±0.7 % 8.9±5.4 % Bortezomib + TRAIL** 23.5±13.5 % 28.6±12.7 %

Description: The study of megakaryocytopoiesis has been based largely on in vitro assays. We characterize an in vivo model of megakaryocyte and platelet development in which human peripheral blood stem cells (PBSCs) differentiate along megakaryocytic as well as myeloid/lymphoid lineages in sublethally irradiated nonobese diabetic/severe combined immunodeficient (NOD-SCID) mice. Human hematopoiesis preferentially occurs in the bone marrow of the murine recipients, and engraftment is independent of exogenous cytokines. Human colony-forming units–megakaryocyte (CFU-MK) develop predominantly in the bone marrow, and their presence correlates with the overall degree of human cell engraftment. Using a sensitive and specific flow cytometric assay, human platelets are detected in the peripheral blood from weeks 1 to 8 after transplantation. The number of circulating human platelets peaks at week 3 with a mean of 20 × 109/L. These human platelets are functional as assessed by CD62P expression in response to thrombin stimulation in vitro. Exogenous cytokines have a detrimental effect on CFU-MK production after 2 weeks, and animals treated with these cytokines have no circulating platelets 8 weeks after transplantation. Although cytokine stimulation of human PBSCs ex vivo led to a significant increase in CFU-MK, CD34+/41+, and CD41+ cells, these ex vivo expanded cells provided only delayed and transient platelet production in vivo, and no CFU-MK developed in vivo after transplantation. In conclusion, xenogeneic transplantation of human PBSCs into NOD/SCID mice provides an excellent in vivo model to study human megakaryocytopoiesis and platelet production.