ALBERT

Description: Human leukocyte antigen class I molecules expressed by tumor cells play a central role in the regulation of natural killer (NK) cell–mediated immune responses. The proteasome inhibitor bortezomib has demonstrated significant activity in multiple myeloma (MM). We hypothesized that treatment of MM with bortezomib results in the reduction of cell-surface expression of class I and thereby sensitizes MM to NK cell–mediated lysis. Here we report that bortezomib down-regulates class I in a time- and dose-dependent fashion on all MM cell lines and patient MM cells tested. Downregulation of class I can also be induced in vivo after a single dose of 1.0 mg/m2 bortezomib. Bortezomib significantly enhances the sensitivity of patient myeloma to allogeneic and autologous NK cell–mediated lysis. Further, the level of decrease in class I expression correlates with increased susceptibility to lysis by NK cells. Clinically relevant bortezomib concentrations do not affect NK-cell function. Our findings have clear therapeutic implications for MM and other NK cell–sensitive malignancies in the context of both allogeneic and autologous adoptively transferred NK cells.

Description: Introduction: Natural killer (NK) cell activity is regulated by a dynamic balance between inhibitory and activating receptors that recognize ligands on target cells. Human leukocyte antigen (HLA)-class I, particularly HLA-C and -Bw4 molecules, are key ligands transmitting inhibitory signals to NK cells. NK cells avidly lyse tumor cells that do not display such inhibitory KIR-ligands. The proteasome is responsible for the generation of peptides that bind to and stabilize class I molecules at the cell surface. We hypothesized that bortezomib, a partial proteasome inhibitor that is clinically approved for the treatment of refractory/relapsed myeloma (MM), could reduce HLA expression on MM cells and thus enhance NK cell-mediated cytotoxicity. Methods: HLA-class I or HLA-C expression was assessed using flow cytometry, after gating on AnnexinV/PI double negative cells, and/or confocal microscopy. Expression of other proteins was measured by flow cytometry using specific mAb. NK cell-mediated lysis of myeloma cells was measured by 51Cr-release. Results: Bortezomib at clinically attainable concentrations down-regulated HLA-class I expression on MM cells in a time- and dose-dependent fashion. Reduction of class I post-10 nM bortezomib treatment was observed in all myeloma cell lines tested (n=10), by a median of 49% (range: 19–66%). A similar decrease of HLA-class I was obtained in 10–50 nM bortezomib treated primary MM cells (n=6). Bortezomib significantly enhanced the sensitivity of MM cells to allogeneic and autologous NK cell-mediated lysis. Further, the level of reduction in HLA-class I expression correlated well with increased susceptibility to lysis by NK cells. The level of down-regulation of HLA-class I induced by bortezomib was reproduced by incubating MM cells with HLA-blocking antibody and resulted in equipotent enhancement of NK cell-mediated lysis (Figure 1). The extent of HLA-class I down-regulation by bortezomib was therefore biologically relevant. Down-regulation of HLA-class I was also observed in vivo on purified MM cells 48 hours after a single dose of bortezomib, by a median of 47% (range: 16–63%, n=6, P= .002). HLA-C expression (the principal NK cell inhibitory ligand) was rescued by exogenous provision of HLA-C binding peptides providing a mechanistic explanation for the effect of bortezomib on HLA-class I expression. Finally, we did not observe bortezomib-mediated enhancement of NK cell-mediated lysis of myeloma through receptors other than the KIR receptor family, including tumor necrosis factor related apoptosis-inducing ligand, NKG2D and natural cytotoxicity receptors. HLA-class I down-regulation was not observed in renal cell and breast carcinoma cell lines, which is in keeping with the remarkable activity of bortezomib in myeloma. Our findings have clear therapeutic implications for MM and other NK cell-sensitive malignancies in the context of both allogeneic and autologous adoptively transferred NK cells. Figure 1. Reduced class I on MM cell surface results in NK cell-mediated recognition and lysis Figure 1. Reduced class I on MM cell surface results in NK cell-mediated recognition and lysis

Description: Background: Tumor cells exhibit elevated ROS levels and cancer stem cells have lower ROS levels than their normal counterparts [Kobayashi et al., 2011]. However, little is known about the effect of ROS levels on multiple myeloma (MM) plasma cell (PC) chemosensitivity. Previously we found that ROS levels are variable in CD138-selected MMPCs from different cases at diagnosis. However, ROS levels in MMPCs from focal lesions (FL, n=42) are lower than PCs from random bone marrow (BM) aspirates (n=192, p=0.02). ROS levels also vary among MM molecular subgroups, with MF and CD2 subgroups having low ROS levels, suggesting that the microenvironment and genetic factors affect their oxidative capacities. We hypothesized that ROS level may reflect the cancer stemness properties of MM cells. We first sought to establish a model to investigate MM cells with different ROS levels and then determine if this property confers cancer stemness and chemoresistance. Methods: We analyzed gene expression profiles (GEP) of primary myeloma cells from newly diagnosed patients with low (n=48) and high (n=48) ROS level by gene set enrichment analysis. We compared viability, proliferation, cell cycle status, drug efflux ability, chemosensitivity, and clonogenic capacity of MM cell lines H929 and U266 with high and low ROS levels, sorted by FACS using H2DCFDA. We also investigated whether MMPCs with low or high ROS levels would differ in their sensitivity to BBI-608, a drug that is reported to target STAT3 driven gene transcription and cancer stemness properties in solid cancers [Li et al., 2015] and is currently being tested in hematologic cancers [BBI608-103HEME]. Results: Pathways enrichment analysis revealed that primary MM cases with low ROS had increased oxidative phosphorylation, proteasome activity, and nucleotide excision repair compared to cases with high ROS that had increased glycolysis, and chemokine and calcium signaling pathways. To explore these differences we separated MMCLs into cells not sorted by ROS level (non-ROS sorted), ROSLo, and ROSHi groups. After sorting, H929 cells retained significant differences in ROS levels for 48-72 hr and U266 cells returned to basal ROS levels within 24hr. ROSLo H929 cells had significantly reduced cell number (p

Description: Introduction The complex makeup of the tumor microenvironment (ME) exerts selective pressure on cancer cells leading to immune escape, and novel immunotherapeutic interventions have emerged from evolving knowledge of the immune system and tumor cells. Natural killer (NK) cells are innate immune cells that exert potent anti-tumor effects. Previously we have reported that ex vivo expansion of NK cells by co-culture PBMCs with K562mbIL15-41BBL can generate large numbers of highly active expanded NK cells (ENKs). These ENKs expand further upon adoptive transfer in vivo both in a murine model and in patients (Garg et al. 2012, Szmania et al. 2015), and have been shown to persist and retain their cytolytic ability. We are currently applying ENK therapy in a Phase II clinical trial at our institute in gene expression profiling-defined high-risk multiple myeloma (MM), a patient population which fares poorly despite the use of novel drugs and autologous stem cell transplantation. A potential obstacle to successful NK cell-based therapies is the suppression of NK cells in the MM bone marrow ME (BM-ME) by immunosuppressive cells, various soluble factors (SF), microRNAs, and exosomes. Exosomes are endosomal-derived, 30-130nm microvesicles present in almost all body fluids. Their number is significantly higher in cancer patients. Tumor-derived exosomes contain a wide range of bioactive molecules, such as microRNA, RNA, DNA and protein, and play a major role in immune escape, promoting tumor progression. Their size, structure, and presence in serum allow them to transport their cargo to distant targets. This study was designed to characterize the potential adverse effects of myeloma-derived exosomes (MEXs) and myeloma-derived SF (MSF) on NK cell function and determine if such inhibition can be overcome by cytokine support. Methods MEXs were isolated from OPM2 myeloma cell line-derived conditioned media (MCM) using the Total Isolation Reagent (Life Technologies, Carlsbad CA). Transmission electron microscopy (TEM), flow cytometry, and western blot (WB) analysis were used for characterization of exosomes. Fresh NK cells (non-activated) and ENKs were incubated with MCM or MEXs and evaluated for their viability and cytolytic ability in standard 4-hour chromium release assays. Flow cytometry was used to evaluate the immunophenotype of these cells, including activation, costimulatory, inhibitory receptors, and adhesion molecules. Results TEM confirmed the presence of exosomes in MCM (size and morphology). Interestingly, OPM2-derived MEXs did not express the exosome-specific marker CD9, but did express CD63, and CD81. Flow cytometry showed that MEXs contain MICA/B, TGFβ, TRAIL-R1, TRAIL-R2, MHC class I, HLA-E, and ICAM3. NK cells exposed to MEXs demonstrated a dose-dependent, significant decrease in specific lysis of the MM cell lines JJN3, OPM2, and U266 in cytotoxicity assays compared to control NK cells (13%-51%, p

Description: Introduction: Immunotherapy with innately competent natural killer (NK) cells can potentially synergize with chemotherapy by eradicating chemo-refractory residual disease. We recently reported that killer immunoglobulin-like receptor (KIR)-ligand mismatched NK cells from haplo-identical donors are cytotoxic to primary myeloma (MM) cells in vitro. In a pilot trial complete remission was achieved in 50% of patients with advanced MM. Although the initial clinical results were encouraging virtually all patients relapsed, suggesting that the number of allo-reactive NK cells transfused was too low to eradicate all MM cells taking into account that MM patients may harbor a tumor burden as large as 109–10 MM cells. This provided the rationale for expanding and activating NK cells from healthy donors with irradiated K562 cells transfected with 4-1BBL and membrane-bound IL15 (K562 transfectants), as described by Imai et al (Blood2005;106:376–383). Methods: PBMNC from 6 healthy donors were co-cultured for 14 days with irradiated K562 cell transfectants. The cultures were then analyzed for fold expansion, gene expression using the Affymetrix U133Plus2.0 microarray platform, immunophenotyping and cytotoxicity. Results: There was an average of 166-fold (range 54–267) expansion of NK cells in the 12 experiments from 6 healthy donors studied after 14 days of co-culture with K562 transfectants without expansion of T-cells. Comparison of gene expression profiles of purified, non-expanded and expanded NK cells of healthy donors showed up-regulation of the expression of activation molecules (e.g. NKG2D, NCRs, DNAM-1), death receptors, granzymes and adhesion molecules on activated/expanded NK cells all consistent with increased proclivity to MM killing. Up regulation of activating receptors was confirmed by flow cytometry. In contrast to non-expanded NK cells, inhibitory KIR receptors were not up regulated on activated NK cells. Using the same number of NK cells, the median lysis of primary MM by expanded NK cells was 62% (range: 49%–70%, n=6) at a 10:1 E:T ratio compared to non-expanded NK cells (median: 23%; range: 15%–34%, p

Description: Abstract 2604 Poster Board II-580 Juvenile myelomonocytic leukemia (JMML) is a mixed myelodysplastic /myeloproliferative neoplasm (MDS/MPN) of infancy and early childhood. The pathogenesis of JMML has been linked to dysregulated signal transduction through the NF1/RAS signaling pathway and PTPN11. This dysregulation results in JMML cells demonstrating selective hypersensitivity to GM-CSF in in vitro dose-response assays. Since JMML hematopoietic progenitor cells are selectively hypersensitive to (rather than independent of) GM-CSF, it is rational to hypothesize that the function of the GM-CSF receptor in JMML patients is not constitutively over-active unless stimulated by the cytokine. We previously reported that PTEN is deficient in JMML patients. PTEN expression is up-regulated by Egr-1, which is one of the targets of the cAMP-response-element-binding protein (CREB). CREB, as a transcriptional factor, is expressed ubiquitously and bound to the cAMP-response-element (CRE) of the Egr-1 promoter. After phosphorylation at serine 133, CREB selectively activates the transcription of Egr-1 in response to GM-CSF stimulation in hematopoietic cells. We evaluated the CREB protein level in peripheral blood or bone marrow samples collected from 26 JMML patients. Mononuclear cells (MNCs) were isolated and lysed in lysis buffer at a density of 107/100μl. Protein levels of CREB were evaluated by ELISA and Western-blot. We found that 22/26 (85%) of subjects were substantially CREB deficient while they had constitutively high activity of MAP kinase (Erk-1/2). In comparison to normal controls (n=7), the median level of total CREB protein by ELISA was significantly lower in JMML subjects (0.62 vs 8.85 ng/mg BSA in normal controls; p=0.006). The mechanism that causes CREB deficiency in JMML is under further investigation and further results may be available to present at the meeting. This is the first evidence that CREB, a critical component downstream of the GM-CSF receptor, is highly deficient in the majority of JMML cases. Disclosures: No relevant conflicts of interest to declare.