scholarly journals Targeting Bclxl Mitigates Mcl1 Chemoresistance in Multiple Myeloma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2656-2656
Author(s):  
Ioanna Savvidou ◽  
Tiffany Khong ◽  
Irena Carmichael ◽  
Jaynish S Shah ◽  
Sridurga Mithraprabhu ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Research Funding ◽  
Ex Vivo ◽  
Combined Treatment ◽  
Live Imaging ◽  
Synergistic Activity ◽  
Protein Levels ◽  
Bax Protein

Abstract Aim: Despite the adoption of novel therapeutic modalities, Multiple Myeloma (MM) remains incurable. The Bcl2 inhibitor Venetoclax is active in several haematologic malignancies, but the benefits in MM patients are limited to those with the t(11;14) and/or high Bcl2 expression. These results underscore the significance of Bcl2 alternative anti-apoptotic proteins (Mcl1 and BclxL) for the survival of myeloma cells. Method: We validated the anti MM effect of the Mcl1 inhibitor S63845 both in vitro utilising 11 human myeloma cell lines (HMCL) and ex vivo against n=30 primary MM tumours. Comparative analysis of RNAseq between S63845 sensitive and resistant HMCL was undertaken to identify candidate proteins that potentially modulate resistance to S63845. Treatment with S63845 and rationally selected combination partners was further evaluated in vitro, ex vivo and in vivo with flow cytometry, immunoblotting and live imaging mitochondria fitness monitoring. Results: RNAseq identified BclxL as potential mediator of resistance to S63845 in HMCL. Immunoblotting confirmed high BclxL expression and high BclxL/BclS in S63845 resistant HMCL. Five S63845 resistant HMCL (U266, ANBL6, KMS28PE, EJM, MM1R) and primary tumours were treated with S63845 combined with the BclxL inhibitor A1331852 . Combined treatment of the HMCL demonstrated a high Bliss synergy score for all the HMCL tested (54, 42, 24, 47, 45 for U266, EJM, KMS28PE, MM1R and ANBL6 respectively) and induced synergistic killing of 80% of the primary tumours treated. Dual inhibition in U266 induced an 80% drop in intracellular ATP at 4h with an increase in active Caspases 9 and 8 (4.5 and 5 fold, respectively). Similarly, the combination induced a 78% drop in mitochondrial transmembrane potential (TMRE intensity) by 4h with live imaging revealing striking mitochondrial damage as early as 40 minutes after exposure (figure). These changes were associated with a reduction of both Mcl1 and, BclxL proteins and Bim and Bid protein levels. No changes were seen in the level of Bcl2, Bak or Bax protein expression. The combination of S63845 and A1331852 in healthy NSG mice at 12.5mg/kg proved lethal due to hepatotoxicity, arguing against the clinical utility of such an approach. However, this observed anti-MM synergistic activity was recapitulated when S63845 was combined with the already approved anti-MM therapeutic panobinostat, with the induction of a significant reduction in both BclxL and Myc protein levels at 24h, and synergistic killing of 56% of primary tumours. Conclusion: High BclxL expression and BclxL/BclxS ratio correlates with resistance to the Mcl1 inhibitor S63845. A combinatorial approach targeting Mcl1 and BclxL induced immediate and significant anti-MM effect both in vitro and ex vivo but proved to be toxic in vivo. Combination of the anti-MM therapeutic panobinostat in combination with S635845 recapitulated the anti-MM activity seen with A1331852 and warrants further evaluation. Figure 1 Figure 1. Disclosures Spencer: Celgene: Honoraria, Research Funding, Speakers Bureau; Janssen: Honoraria, Research Funding, Speakers Bureau; Amgen: Honoraria, Research Funding; Bristol Myers Squibb: Research Funding; Takeda: Honoraria, Research Funding, Speakers Bureau; STA: Honoraria.

Towards CD38-Targeted Immunochemotherapy of Multiple Myeloma: Significant Improvement of Anti-Myeloma Effects of Lenalidomide, Bortezomib and Dexamethason by CD38-Specific Antibody Daratumumab, Even in Chemotherapy Resistant/Intolerant Patients

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4988-4988
Author(s):  
Inger S. Nijhof ◽  
Jeroen Lammerts van Bueren ◽  
Berris van Kessel ◽  
Michel de Weers ◽  
Joost M Bakker ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Research Funding ◽  
Plasma Cells ◽  
Ex Vivo ◽  
Human Antibody ◽  
Synergistic Activity ◽  
Effector Cells ◽  
Complement Dependent Cytotoxicity

Abstract Abstract 4988 To date, multiple myeloma (MM) remains an incurable malignancy of antibody-producing clonal plasma cells. The introduction of a new generation of immunomodulatory agents, such as lenalidomide (LEN), and the potent proteasome inhibitor bortezomib (BORT), used alone or in combination with steroids (dexamethasone; DEX or prednisone; PRED) has significantly improved the overall survival of MM patients. Nonetheless, all chemotherapy strategies are eventually hampered by the development of drug-resistance. Towards a novel and effective targeted immunotherapy for MM, we have developed daratumumab (DARA), a CD38 human antibody with broad-spectrum killing activity. In vitro, DARA induces substantial anti-MM effects mainly via ADCC (antibody dependent cellular cytotoxicity) and CDC (complement dependent cytotoxicity). In ex vivo assays, which allowed us to address killing of MM cells in bone marrow aspirates isolated from MM patients, enhanced or even synergistic MM cell killing was observed when DARA was combined with LEN, or with cocktails of LEN/BORT/DEX and melphalan/BORT/DEX. We now extended these ex vivo analyses to evaluate whether DARA in combination with LEN, BORT and DEX could improve the lysis of MM cells in bone marrow aspirates derived from 22 patients of whom 9 became refractory for LEN and 6 for LEN and BORT. DARA significantly enhanced the lysis of MM cells when combined with LEN or BORT in virtually all patients, including the LEN- and LEN/BORT-refractory patients. The combination of DARA+BORT and DARA+DEX induced additive killing, suggestive of lysis by independent mechanisms. When combined with LEN, DARA improved the lysis of MM cells in a synergistic manner in both non-refractory and LEN-refractory patients. This is suggestive of killing by at least partly complementary mechanisms. Synergistic activity of LEN and DARA was attributable to LEN-induced activation of effector cells that were involved in DARA-mediated ADCC. In addition, enhanced/synergistic direct killing of MM cells was observed. Experiments are under way to further investigate the mechanism underlying synergistic activity of DARA and LEN. In conclusion, our results provide a rationale for clinical evaluation of DARA in combination with LEN, BORT and DEX including in patients refractory to these drugs. Disclosures: van Bueren: genmab: Employment. de Weers:genmab: Employment. Bakker:genmab: Employment. Parren:genmab: Employment. Lokhorst:genmab: Consultancy, Research Funding. Mutis:genmab: Research Funding.

scholarly journals Hypoxia-induced CREB cooperates MMSET to modify chromatin and promote DKK1 expression in multiple myeloma

Oncogene ◽  
2021 ◽  
Author(s):  
Yinyin Xu ◽  
Jing Guo ◽  
Jing Liu ◽  
Ying Xie ◽  
Xin Li ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Combined Treatment ◽  
Hypoxia Inducible Factor ◽  
Bone Destruction ◽  
Bone Lesions ◽  
Myeloma Cells ◽  
Downstream Target ◽  
Dkk1 Expression

AbstractMyeloma cells produce excessive levels of dickkopf-1 (DKK1), which mediates the inhibition of Wnt signaling in osteoblasts, leading to multiple myeloma (MM) bone disease. Nevertheless, the precise mechanisms underlying DKK1 overexpression in myeloma remain incompletely understood. Herein, we provide evidence that hypoxia promotes DKK1 expression in myeloma cells. Under hypoxic conditions, p38 kinase phosphorylated cAMP-responsive element-binding protein (CREB) and drove its nuclear import to activate DKK1 transcription. In addition, high levels of DKK1 were associated with the presence of focal bone lesions in patients with t(4;14) MM, overexpressing the histone methyltransferase MMSET, which was identified as a downstream target gene of hypoxia-inducible factor (HIF)-1α. Furthermore, we found that CREB could recruit MMSET, leading to the stabilization of HIF-1α protein and the increased dimethylation of histone H3 at lysine 36 on the DKK1 promoter. Knockdown of CREB in myeloma cells alleviated the suppression of osteoblastogenesis by myeloma-secreted DKK1 in vitro. Combined treatment with a CREB inhibitor and the hypoxia-activated prodrug TH-302 (evofosfamide) significantly reduced MM-induced bone destruction in vivo. Taken together, our findings reveal that hypoxia and a cytogenetic abnormality regulate DKK1 expression in myeloma cells, and provide an additional rationale for the development of therapeutic strategies that interrupt DKK1 to cure MM.

scholarly journals A novel BCMA/CD3 bispecific T-cell engager for the treatment of multiple myeloma induces selective lysis in vitro and in vivo

Leukemia ◽  
2016 ◽  
Vol 31 (8) ◽  
pp. 1743-1751 ◽  
Author(s):  
S Hipp ◽  
Y-T Tai ◽  
D Blanset ◽  
P Deegen ◽  
J Wahl ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
T Cell ◽  
Plasma Cells ◽  
Ex Vivo ◽  
Xenograft Model ◽  
Selective Lysis ◽  
Bispecific T Cell Engager

Abstract B-cell maturation antigen (BCMA) is a highly plasma cell-selective protein that is expressed on malignant plasma cells of multiple myeloma (MM) patients and therefore is an ideal target for T-cell redirecting therapies. We developed a bispecific T-cell engager (BiTE) targeting BCMA and CD3ɛ (BI 836909) and studied its therapeutic impacts on MM. BI 836909 induced selective lysis of BCMA-positive MM cells, activation of T cells, release of cytokines and T-cell proliferation; whereas BCMA-negative cells were not affected. Activity of BI 836909 was not influenced by the presence of bone marrow stromal cells, soluble BCMA or a proliferation-inducing ligand (APRIL). In ex vivo assays, BI 836909 induced potent autologous MM cell lysis in both, newly diagnosed and relapsed/refractory patient samples. In mouse xenograft studies, BI 836909 induced tumor cell depletion in a subcutaneous NCI-H929 xenograft model and prolonged survival in an orthotopic L-363 xenograft model. In a cynomolgus monkey study, administration of BI 836909 led to depletion of BCMA-positive plasma cells in the bone marrow. Taken together, these results show that BI 836909 is a highly potent and efficacious approach to selectively deplete BCMA-positive MM cells and represents a novel immunotherapeutic for the treatment of MM.

scholarly journals Negative Impact of Hypoxia on Tryptophan 2,3-Dioxygenase Function

2016 ◽  
Vol 2016 ◽  
pp. 1-11 ◽  
Author(s):  
Frank Elbers ◽  
Claudia Woite ◽  
Valentina Antoni ◽  
Sara Stein ◽  
Hiroshi Funakoshi ◽  
...  
Keyword(s):  
Essential Amino Acid ◽  
Negative Impact ◽  
Ex Vivo ◽  
T Cell Proliferation ◽  
Effector Functions ◽  
Protein Levels ◽  
Hypoxic Conditions ◽  
Oxygen Conditions

Tryptophan is an essential amino acid for hosts and pathogens. The liver enzyme tryptophan 2,3-dioxygenase (TDO) provokes, by its ability to degrade tryptophan to N-formylkynurenine, the precursor of the immune-relevant kynurenines, direct and indirect antimicrobial and immunoregulatory states. Up to now these TDO-mediated broad-spectrum effector functions have never been observed under hypoxiain vitro, although physiologic oxygen concentrations in liver tissue are low, especially in case of infection. Here we analysed recombinant expressed human TDO andex vivomurine TDO functions under different oxygen conditions and show that TDO-induced restrictions of clinically relevant pathogens (bacteria, parasites) and of T cell proliferation are abrogated under hypoxic conditions. We pinpointed the loss of TDO efficiency to the reduction of TDO activity, since cell survival and TDO protein levels were unaffected. In conclusion, the potent antimicrobial as well as immunoregulatory effects of TDO were substantially impaired under hypoxic conditions that pathophysiologically occurin vivo. This might be detrimental for the appropriate host immune response towards relevant pathogens.

The Novel Aurora Kinase Inhibitor ENMD-2076 Has Potent Single Agent Activity against Multiple Myeloma (MM) in Vitro and in Vivo, and Shows Synergistic Activity in Combination with Lenalidomide

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3660-3660 ◽  
Author(s):  
Xiaojing Wang ◽  
Anthony L. Sinn ◽  
Attaya Suvannasankha ◽  
Colin D. Crean ◽  
Li Chen ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Caspase 3 ◽  
Kinase Inhibitor ◽  
Single Agent ◽  
Synergistic Activity ◽  
Significant Activity ◽  
Aurora A Kinase ◽  
Free Base

Abstract ENMD-2076 is a novel, orally-active molecule that has been shown to have significant activity against Aurora A kinase as well as multiple receptor tyrosine kinases (RTK). We investigated the single agent activity of ENMD-2076 against MM cells in vitro and in vivo, and in combination with lenalidomide. ENMD-2076 free base showed significant cytotoxicity against MM cells with a mean LC50 of 3.84±0.86 μM at 48 hours in vitro. Cytotoxicity was associated with cleavage of caspase 3, 8, 9 and PARP, and loss of mitochondrial membrane potential as early as 6 hours. ENMD-2076 free base inhibited c-kit, FGFR-1, 3 and VEGFR1 and subsequently inhibition of downstream targets phosphorylated (p)-BAD, p-Foxo1a and p-GSK-3β was observed at 6 hours. NOD/SCID mice implanted with H929 human plasmacytoma xenografts and treated for 30 days with 50, 100, 200mg/kg/d ENMD-2076 showed a dose-dependent inhibition of tumor growth (Figure 1), with minimal toxicity as assessed by the stable weight of treated animals. Immunohistochemical staining of tumors from sacrificed animals showed significant reduction in Ki67 at all dose levels of treatment compared to control tumors. An increase in cleaved caspase-3 was observed on Western blot from the lysates of H929 tumors obtained from treated animals. ENMD-2076 free base also showed synergistic cytotoxic activity when combined with lenalidomide against H929, MM1.R and MM1.S cells as assessed by MTT assay and Annexin-V/PI staining. Using the Chou-Talalay method, the combination indices (CI) were < 1 for all three cell lines across a range of concentrations of ENMD-2076 free base (0.25–1.0 μM) plus lenalidomide (2.5–10 μM) indicating synergistic activity (CI=0.362 H929; CI=0.315 MM1.R; CI=0.415 MM1.S). Our results provide rationale for the investigation of ENMD-2076 alone and in combination with lenalidomide in patients with multiple myeloma. Figure Figure

Cytokine-Mediated Signaling Is Suppressed in Myeloid Cells and Enhanced in Lymphatic Cells in Patients with Chronic Myeloid Leukemia (CML) — Partial Normalization with Tyrosine Kinase Inhibitors.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2180-2180
Author(s):  
Sari Jalkanen ◽  
Satu Mustjoki ◽  
Kimmo Porkka ◽  
Jukka Vakkila
Keyword(s):  
T Cells ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Ex Vivo ◽  
Healthy Controls ◽  
Gm Csf ◽  
Single Cell Profiling ◽  
Treg Lymphocytes

Abstract Abstract 2180 Poster Board II-157 Introduction. Aberrant phosphorylation of the BCR-ABL1 tyrosine kinase (TK) is characteristic of chronic myeloid leukemia (CML). This oncoprotein interacts directly with intracellular signaling proteins, alters the responsiveness of cytokine receptors and regulates secretion of autocrine cytokines. Targeted inhibition of BCR-ABL1 with TK inhibitor (TKI) imatinib mesylate (IM) is the current standard treatment of CML. For overcoming IM resistance or intolerance, 2nd generation TKIs (nilotinib, dasatinib) with broader kinase inhibition profile have been approved for clinical use. Although in vitro results suggest that TKIs are immunosuppressive, no increases in opportunistic infections or secondary malignancies have been observed to date. In contrast, in some TKI-treated patients immunoactivation in the form of chronic lymphocytosis linked to excellent therapy responses has recently been shown. Dynamic monitoring of aberrant cytokine signaling pathways would aid in understanding and predicting the development of TKI-resistance or adverse/off-target effects. The aim of this study was to analyze the responsiveness of leukocytes to cytokine stimuli in CML patients at diagnosis and during TKI therapy using single-cell profiling of phosphoprotein networks by multiparameter flow cytometry. Patients and methods. The study consisted of 4 healthy controls, 6 CML patients at diagnosis, 6 IM patients and 5 dasatinib patients. Stimuli included GM-CSF, IL-2+IL-10+IFNα and IL-4+IL-6+IFNγ and they were added immeadately to freshly drawn whole blood ex vivo. The readout phosphoproteins were pERK1/2, pSTAT1, pSTAT3, pSTAT5a and pSTAT6 (with isotype controls), and were analyzed separately from granulocytes, monocytes, CD4+ CD25neg T helper cells (Th), CD4neg lymphocytes and CD4+CD25+ T cells including regulatory T-cells (Treg). Analysis was performed with heatmap function of Cytobank software (http://cytobank.stanford.edu/public/). Results. Unstimulated phosphoprotein levels reflecting the activation state of leukocytes in vivo did not differ between healthy controls and CML patients at diagnosis or during dasatinib therapy. Strikingly, in IM patients, baseline levels of pSTAT3 were relatively high indicating in vivo occurring activation of leukocytes in this patient group. We next studied ex vivo responsiveness of immune effector cells with cytokines and found clear differences between healthy controls and CML patients. At CML diagnosis. GM-CSF/pERK1+pSTAT5a, IFNa/pSTAT1,and IL-4/pSTAT6 (stimulus/readout) as well as pSTAT3 responses with all stimuli were suppressed in monocytes. In granulocytes, GM-CSF/pSTAT1 levels were diminished. In Th and Treg lymphocytes, IL-6/pSTAT3 responses were markedly pronounced, while IL-10/pSTAT3 responses were not affected when compared to healthy controls. Such difference was not observed in CD4neg lymphocytes. During TKI therapy. Most patients (9/11) were in cytogenetic remission at the time of analysis. The unresponsiveness of myeloid cells at diagnosis was restored by IM or dasatinib therapy in most, but not all patients. Similarly, in Th and Treg lymphocytes TKI-therapy normalized the enhanced IL-6/pSTAT3 responses that were evident at diagnosis. However, in Th and Treg cells pSTAT3 responses provoked by IL-10 were particularly prominent. Interestingly, one dasatinib patient with aberrant constant blood NK-lymphocytosis and monocytosis had uniquely strong IFNg/pSTAT1 and IL-4/pSTAT6 responses in monocytes. Furthermore, one patient who have stayed in persistent remission after IM discontinuation had exceptionally high pSTAT3 responses with all of stimuli used. Similar kind of signaling profile was unseen with the other patients and could reflect immunoactivation related to leukemia control. Conclusions. Dynamic single-cell profiling of signaling networks is feasible in CML patients and can be used to study mechanisms of aberrant immune reactivity in TKI-treated patients. The method could be particularly suitable for assessing candidate patients for TKI discontinuation. Although in vitro results suggest immunosuppressive effects of TKIs on lymphocytes, leukocytes ex vivo from patients were able to respond similarly to cytokine stimuli as in healthy controls. Disclosures: Mustjoki: BMS: Honoraria. Porkka:BMS: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.

Pralatrexate Has Potent Activity Against Multiple Myeloma In Vitro and In Vivo, and Activity Correlates with Tumor RFC-1 and DHFR Expression

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1831-1831 ◽  
Author(s):  
Michael Mangone ◽  
Luigi Scotto ◽  
Enrica Marchi ◽  
Owen A. O'Connor ◽  
Hearn J. Cho
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Research Funding ◽  
Folate Metabolism ◽  
Nog Mice ◽  
Induced Apoptosis ◽  
Dose Dependent ◽  
Functional Biomarkers

Abstract Abstract 1831 Multiple myeloma (MM) is the second most common hematologic malignancy. Although there are effective new agents that can induce remission, relapse is inevitable and the disease is currently incurable. Progress in the treatment of this disease demands development of novel therapeutics and identification of functional biomarkers that may be used to distinguish tumors that are susceptible to specific targeted agents, creating a “personalized” therapeutic strategy for individual patients. We investigated these principles with anti-folates, which are not commonly used in MM but have demonstrated activity in this disease. Pralatrexate (PDX, 10-propargyl 10-deazaaminopterin) is a folate analogue that was rationally designed to have high affinity for Reduced Folate Carrier (RFC)-1, an oncofetal protein expressed in many cancers that actively transports folates into cells. PDX induced dose-dependent apoptotic cell death in a subset of human myeloma cell lines (HMCL) and CD138+ MM cells isolated from a clinical specimen. In sensitive cell lines, PDX exhibited 10-fold greater potency compared to the structurally related drug methotrexate (MTX). PDX induced dose-dependent, intrinsic apoptosis in sensitive HMCLs, characterized by cleavage of caspase-3 and -9 and accompanied by the loss of full-length Mcl-1, a Bcl-2 family protein that plays a critical role in drug-induced apoptosis in MM. Furthermore, the activity of PDX is not abrogated by the presence of exogenous interleukin-6 or by co-culture with HS-5 bone marrow stromal cells, both of which exert powerful survival effects on MM cells and can antagonize apoptosis in response to some cytotoxic chemotherapy drugs. Sensitivity to PDX-induced apoptosis correlated with higher relative levels of RFC-1 mRNA in sensitive compared to resistant HMCL. Resistant HMCL also exhibited a dose-dependent up-regulation of dihydrofolate reductase (DHFR) protein, a primary molecular target for anti-folates, in response to PDX exposure, whereas sensitive HMCL did not. These changes in functional folate metabolism biomarkers, high baseline RFC-1 expression and upregulation of DHFR in response to PDX, appeared to be mutually exclusive to sensitive or resistant HMCL, respectively. Importantly, PDX was also effective against sensitive HMCL in vivo in a novel mouse xenograft model. NOD/Shi-scid/IL-2Rγnull (NOG) mice were inoculated with MM.1s HMCL stably transduced to express both GFP and luciferase (GFP-luc). GFP-luc MM.1s cells engrafted into the long bones, pelvis, and vertebral column of NOG mice within 4–7 days after injection of cells, as assessed by in vivo bioluminescent imaging. Treatment with PDX resulted in a significant reduction in tumor burden after two doses. These results demonstrate that PDX has potent anti-myeloma activity in vitro and in vivo, and that RFC-1 expression and DHFR upregulation are robust functional biomarkers that may identify patients who are likely to benefit from PDX therapy. These data support further exploration of PDX therapy in clinical trials for MM and investigation of folate metabolism biomarkers as indices for treatment with this class of drugs. Improved anti-folates such as PDX are a promising class of agents that may be a valuable addition to the arsenal against MM. Disclosures: O'Connor: Celgene: Consultancy, Research Funding; Merck: Research Funding; Novartis: Research Funding; Spectrum: Research Funding.

Stroma-Free Ex Vivo Expanded, CD34+ Cord Blood-Derived NK Cells Retain Lytic Activity After Long-Term Engraftment in NSGS Mice

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 580-580
Author(s):  
Mark Wunderlich ◽  
Mahesh Shrestha ◽  
Lin Kang ◽  
Eric Law ◽  
Vladimir Jankovic ◽  
...  
Keyword(s):  
Nk Cells ◽  
Research Funding ◽  
Nk Cell ◽  
Ex Vivo ◽  
Employment Equity ◽  
Cell Product ◽  
Equity Ownership ◽  
Cellular Therapeutics

Abstract Abstract 580 Generating a large number of pure, functional immune cells that can be used in human patients has been a major challenge for NK cell-based immunotherapy. We have successfully established a cultivation method to generate human NK cells from CD34+ cells isolated from donor-matched cord blood and human placental derived stem cells, which were obtained from full-term human placenta. This cultivation method is feeder-free, based on progenitor expansion followed by NK differentiation supported by cytokines including thrombopoietin, stem cell factor, Flt3 ligand, IL-7, IL-15 and IL-2. A graded progression from CD34+ hematopoietic progenitor cells (HSC) to committed NK progenitor cells ultimately results in ∼90% CD3-CD56+ phenotype and is associated with an average 10,000-fold expansion achieved over 35 days. The resulting cells are CD16- and express low level of KIRs, indicating an immature NK cell phenotype, but show active in vitro cytotoxicity against a broad range of tumor cell line targets. The in vivo persistence, maturation and functional activity of HSC-derived NK cells was assessed in NSG mice engineered to express the human cytokines SCF, GM-CSF and IL-3 (NSGS mice). Human IL-2 or IL-15 was injected intraperitoneally three times per week to test the effect of cytokine supplementation on the in vivo transferred NK cells. The presence and detailed immunophenotype of NK cells was assessed in peripheral blood (PB), bone marrow (BM), spleen and liver samples at 7-day intervals up to 28 days post-transfer. Without cytokine supplementation, very few NK cells were detectable at any time-point. Administration of IL-2 resulted in a detectable but modest enhancement of human NK cell persistence. The effect of IL-15 supplementation was significantly greater, leading to the robust persistence of transferred NK cells in circulation, and likely specific homing and expansion in the liver of recipient mice. The discrete response to IL-15 versus IL-2, as well as the preferential accumulation in the liver have not been previously described following adoptive transfer of mature NK cells, and may be unique for the HSC-derived immature NK cell product. Following the in vivo transfer, a significant fraction of human CD56+ cells expressed CD16 and KIRs indicating full physiologic NK differentiation, which appears to be a unique potential of HSC-derived cells. Consistent with this, human CD56+ cells isolated ex vivo efficiently killed K562 targets in in vitro cytotoxicity assays. In contrast to PB, spleen and liver, BM contained a substantial portion of human cells that were CD56/CD16 double negative (DN) but positive for CD244 and CD117, indicating a residual progenitor function in the CD56- fraction of the CD34+ derived cell product. The BM engrafting population was higher in NK cultures at earlier stages of expansion, but was preserved in the day 35- cultured product. The frequency of these cells in the BM increased over time, and showed continued cycling based on in vivo BrdU labeling 28 days post-transfer, suggesting a significant progenitor potential in vivo. Interestingly, DN cells isolated from BM could be efficiently differentiated ex vivo to mature CD56+CD16+ NK cells with in vitro cytotoxic activity against K562. We speculate that under the optimal in vivo conditions these BM engrafting cells may provide a progenitor population to produce a mature NK cell pool in humans, and therefore could contribute to the therapeutic potential of the HSC-derived NK cell product. The in vivo activity of HSC-derived NK cells was further explored using a genetically engineered human AML xenograft model of minimal residual disease (MRD) and initial data indicates significant suppression of AML relapse in animals receiving NK cells following chemotherapy. Collectively, our data demonstrate the utility of humanized mice and in vivo xenograft models in characterizing the biodistribution, persistence, differentiation and functional assessment of human HSC-derived cell therapy products, and characterize the potential of HSC-derived NK cells to be developed as an effective off-the-shelf product for use in adoptive cell therapy approaches in AML. Disclosures: Wunderlich: Celgene Cellular Therapeutics: Research Funding. Shrestha:C: Research Funding. Kang:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Law:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Jankovic:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Zhang:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Herzberg:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Abbot:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Hariri:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Mulloy:Celgene Cellular Therapeutics: Research Funding.

CD38-Targeted Immunochemotherapy Of Multiple Myeloma: Preclinical Evidence For Its Combinatorial Use In Lenalidomide and Bortezomib Refractory/Intolerant MM Patients

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 277-277 ◽  
Author(s):  
Inger S. Nijhof ◽  
Willy A. Noort ◽  
Jeroen Lammerts van Bueren ◽  
Berris van Kessel ◽  
Joost M. Bakker ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Research Funding ◽  
Plasma Cells ◽  
Cell Lysis ◽  
In Vitro Assays ◽  
Human Antibody ◽  
Myeloma Cells ◽  
Clinical Phase

Abstract Multiple myeloma (MM) remains an incurable malignancy of clonal plasma cells. Although the new generation of immunomodulatory agents, such as lenalidomide (LEN), and the potent proteasome inhibitor bortezomib (BORT) have significantly improved the overall survival of MM patients, all chemotherapy strategies are eventually hampered by the development of drug-resistance. The outcome of patients who are refractory to thalidomide, lenalidomide (LEN) and bortezomib (BORT) is very poor. Set out with the idea that targeted immunotherapy with human antibodies may offer new perspectives for MM patients, we have recently developed daratumumab (DARA), a CD38 human antibody with broad-spectrum killing activity, mainly via ADCC (antibody dependent cellular cytotoxicity) and CDC (complement dependent cytotoxicity). In our previous preclinical studies and in current clinical phase I/II trials, DARA induces marked anti-MM activity. Based on these encouraging results, we now explored the potential activity of DARA for patients who are refractory to LEN- and/or BORT. In a recently developed human-mouse hybrid model that allows the in vivo engraftment and outgrowth of patient-derived primary myeloma cells in immune deficient Rag2-/-gc-/- mice, single dose DARA treatment appeared to effectively inhibit the malignant expansion of primary MM cells derived from a LEN- and BORT-refractory patient, indicating the potential efficacy of DARA even in LEN/BORT refractory patients. To substantiate the conclusions of these in vivo data, we conducted in vitro assays, in which full BM-MNCs from LEN (n=11) and LEN/BORT (n=8) refractory patients were treated with DARA alone or the combination of DARA with LEN or BORT to induce MM cell lysis. As expected, LEN alone induced no or little lysis of MM cells in the LEN-refractory patients and also BORT was not able to induce any lysis in the BORT-refractory patients. On the contrary, DARA induced substantial levels of MM cell lysis in all LEN and LEN/BORT-refractory patients. This lysis was significantly enhanced by combination with LEN or BORT. The combination of DARA and BORT improved MM lysis by additive mechanisms. However, LEN improved DARA-mediated lysis of MM cells in a synergistic manner through the activation of effector cells involved in DARA-mediated ADCC. In conclusion, our results demonstrate that DARA is also effective against multiple myeloma cells derived from LEN- and BORT-refractory patients. Especially LEN seems to improve responses in a synergistic manner. Our results provide a rationale for clinical evaluation of DARA in combination with LEN to achieve more effective results in LEN- and BORT-refractory patients. Disclosures: Lammerts van Bueren: Genmab: Employment. Bakker:Genmab: Employment. Parren:Genmab: Employment. van de Donk:Celgene: Research Funding. Lokhorst:Genmab A/S: Consultancy, Research Funding; Celgene: Honoraria; Johnson-Cilag: Honoraria; Mudipharma: Honoraria.

Constitutive and Immunoproteasome Inhibitors Increase IκB and Decrease NF-κB Expression In Dendritic Cell

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3493-3493
Author(s):  
Ahmad-Samer Samer Al-Homsi ◽  
Zhongbin Lai ◽  
Tara Sabrina Roy ◽  
Niholas Kouttab
Keyword(s):  
Multiple Myeloma ◽  
T Cell ◽  
Cell Lines ◽  
Mechanism Of Action ◽  
Peripheral Blood ◽  
Research Funding ◽  
Myeloma Cell ◽  
Multiple Myeloma Cell

Abstract Introduction Constitutive and immunoproteasome inhibitors (C&IPI) were thought to suppress nuclear factor-κB (NF-κB) pathway by preventing IκB degradation, which prevents NF-κB translocation into the nucleus. This mechanism of action has since been questioned by a number of studies. First, bortezomib promoted constitutive NF-κB activity in endothelial cell carcinoma. Second, NF-κB constitutive activity was resistant to bortezomib in multiple myeloma cell lines. Third, bortezomib increased IκB mRNA but post-transcriptionally downregulated IκB in normal cells and in multiple myeloma cell lines resulting in induced canonical NF-κB activation. Lastly, bortezomib increased nuclear levels of IκB as opposed to lowering cytoplasmic levels in cutaneous T cell lymphoma cell line suggesting that nuclear translocation of IκB was possibly responsible for NF-κB inhibition. The inhibitory activity of C&IPI on dendritic cells (DC) is of interest in the prevention of graft versus host disease (GvHD). It has been shown that different C&IPI impede DC maturation and T cell priming both in vitro and in vivo. Herein we sought to understand the mechanism of action of proteasome and immunoproteasome inhibitors on DC and to test their effect on IκB and NF-IκB expression. Materials and Methods We first performed RT PCR on lysates of DC obtained from the peripheral blood of 7 patients who received post-transplant cyclophosphamide and bortezomib as prevention of GvHD on a phase I clinical trial. Patients received allogeneic transplantation from matched-related or unrelated donors. Patients received no other immunosuppressive therapy except for rabbit anti-thymocyte globulin for those receiving graft from unrelated donor. Steroids were not allowed on the study. Samples were obtained on days +1, +4, and +7. The results were analyzed in comparison to samples obtained on day 0 before stem cell infusion. We then performed the same experiment on lysates of DC obtained from the peripheral blood of healthy volunteer donors. DC were untreated or incubated with bortezomib (10 nM for 4 h), carfilzomib (30 nM for 1 h), oprozomib (100 nM and 300 nM for 4 h), ONX 0914 (200 nM for 1 h), PR-825 (125 nM for 1 h), or PR-924 (1000 nM for 1 h). The drug concentration and duration of exposure were chosen based on the IC50 on proteasome activity and to reproduce in vivo conditions. We also performed IκB western blot on DC isolated from peripheral blood of healthy volunteers, untreated or incubated with bortezomib (10 nM for 4 h) or oprozomib (300 nM for 4 h). Each experiment was performed at least in triplicate. Results We found that the combination of cyclophosphamide and bortezomib significantly and progressively increased IκB mRNA while decreasing NF-κB mRNA in DC studied ex vivo. We also found that all studied C&IPI increased IκB mRNA to a variable degree while only oprozomib (300 nM) decreased NF-κB mRNA in DC in vitro. Finally, both bortezomib and oprozomib increased IκB protein level in DC in vitro (figure). Conclusion Our data suggest that C&IPI increase IκB expression in DC. As opposed to the previously reported data in other cell types, the effect is not associated with post-transcriptional downregulation. Cyclophosphamide and bortezomib also decrease NF-κB expression in DC in vivo while only oprozomib had the same effect in vitro. The effect of C&IPI on IκB and NF-κB expression may represent a new mechanism of action and suggests their effect may be cell-type dependent. Disclosures: Al-Homsi: Millennium Pharmaceuticals: Research Funding. Off Label Use: The use of cyclophosphamide and bortezomib for GvHD prevention. Lai:Millennium Pharmaceuticals: Research Funding.

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