scholarly journals Blockade of Tigit on AML-Derived M2 Macrophages Results in Reprograming into the M1 Phenotype and Enhances CD47-Mediated Phagocytosis

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3351-3351
Author(s):  
Franziska Brauneck ◽  
Brit Fischer ◽  
Jasmin Wellbrock ◽  
Carsten Bokemeyer ◽  
Julian Schulze zur Wiesch ◽  
...  
Keyword(s):  
Cell Lines ◽  
Research Funding ◽  
Clinical Parameters ◽  
Intermediate Risk ◽  
M2 Macrophages ◽  
Macrophage Population ◽  
Healthy Donors ◽  
Local Immunosuppression ◽  
M1 Phenotype

Abstract Background: Bidirectional interactions between the tumor microenvironment (TME) and AML cells lead to disease progression through induction of angiogenesis, migration, cancer stemness and local immunosuppression. Leukemia-associated macrophages (LAM) constitute an important cell population within the TME, but little is known about the phenotype, function, and plasticity of these cells. In the present study we provide an extensive characterization of the macrophage population in patients with AML. Methods: The phenotype and expression of co-regulatory receptors was assessed on different bone marrow-derived CD68 +CD14 + LAM populations, in comparison to corresponding CD3 + T-cells and CD117 +CD34 + AML cells (n=35), as well as peripheral blood monocytes from healthy donors (HD, n=16) using multi-parameter flow cytometry. The expression of surface markers and the distribution of LAM subpopulations was correlated with clinical parameters. The effect of a blocking anti-TIGIT antibody on the in vitro plasticity on primary LAMs and monocyte-derived macrophages from healthy donors was investigated. Furthermore, we analyzed if the treatment with blocking anti-TIGIT and anti-CD47 antibodies could increase the anti-leukemic phagocytosis of AML cell lines and in vitro polarized monocyte-derived M2 macrophages. Results: Phenotypic analysis of M1 and M2 macrophages in AML and HD revealed that the predominant macrophage population in patients with AML is made up of immunosuppressive alternatively activated M2 LAMs defined by expression of CD163 and CD86 (M1 AML vs. HD p<0.01 and M2 AML vs. HD p=0.02). These M2 LAMs contained significantly higher frequencies of cells expressing the immune checkpoint receptors TIGIT and TIM-3 than M1 LAMs (TIGIT + M2 vs. M1 p<0.01 and TIM-3 + M2 vs. M1 p<0.01, respectively). Regarding co-expression of multiple co-inhibitory receptors, the frequency of macrophages co-expressing TIM-3 or LAG-3 with TIGIT was higher in samples from AML patients in comparison to HDs (p=0.01 and p<0.01, respectively). This difference was caused by the significant up-regulation of TIM-3 and LAG-3 on TIGIT + M2 LAMs in comparison to their corresponding M1 LAMs (p<0.01and p<0.01, respectively). Importantly, in vitro blockade of TIGIT in primary LAMs of AML patients or differentiated PB-derived M2 macrophages of HDs resulted in a change in polarization from the M2 towards the M1 phenotype after 24 hours (AML: anti-TIGIT vs. IgG2a p<0.01, n=7 and HD: anti-TIGIT vs. IgG2a p=0.02, n=3). Moreover, the additional blockade of TIGIT on PB-derived M2 macrophages augmented the anti-CD47-mediated phagocytosis of the AML cell lines MOLM-13 and MV4-11 after 4 hours (MOLM-13: anti-CD47 vs. IgG1a 31% vs. 10.9%, p=0.04; anti-CD47 vs. combined anti-CD47 + anti-TIGIT 31% vs. 46.4%, p<0.01 and combined anti-CD47 + anti-TIGIT vs. IgG1a + IgG2a 46.4% vs. 13.6%, p<0.01, n=3 and for MV4-11: anti-CD47 vs. IgG1a 14.4% vs. 7.345%, p=0.03; anti-CD47 vs. combined anti-CD47 + anti-TIGIT 14.4% vs. 28.6%, p=0.03 and combined anti-CD47 + anti-TIGIT vs. IgG1a + IgG2a 28.6% vs. 12.85%, p=0.04, n=2). Next, we correlated the phenotypic data with clinical parameters. AML patients of the intermediate risk group according to ELN criteria exhibited a significantly higher frequency of M2 LAMs co-expressing TIGIT and LAG-3 than those in the favorable group (p=0.04 and p=0.01). Moreover, the frequency of TIM-3 + M2 LAMs was significantly increased in patients with adverse and intermediate risk in comparison to those with a favorable risk (p=0.01, p=0.0053). Furthermore, TIGIT + M2 LAMs were significantly more frequent in patients with the FLT3 ITD mutation in comparison with the wilde type (p=0.03). Conclusions: Our findings suggest that the proven clinical effect of monoclonal antibodies against TIGIT and TIM-3 in cancer may be due in part to their action on macrophages and depend on macrophage polarization. Our study identifies TIGIT + M2 LAMs co-expressing TIM-3 and LAG-3 as a promising effector population in AML. Further experiments should be conducted to investigate macrophage-mediated cytotoxicity in AML. Disclosures Brauneck: Daiichi Sankyo: Consultancy, Honoraria, Other: meeting attendance; Servier: Consultancy, Honoraria, Other: meeting attendance; Jazz Pharmaceuticals: Other: meeting attendance; Novartis: Other: meeting attendance. Bokemeyer: BMS: Honoraria, Other: Travel accomodation, Research Funding; Sanofi: Consultancy, Honoraria, Other: Travel accomodation; Merck Serono: Consultancy, Other: Travel accomodation ; Bayer Schering Pharma: Consultancy; GSO: Consultancy; AOK Health insurance: Consultancy; Abbvie: Research Funding; ADC Therapeutics: Research Funding; Agile Therapeutics: Research Funding; Alexion Pharmaceuticals: Research Funding; Amgen: Research Funding; Apellis Pharmaceuticals: Research Funding; Astellas: Research Funding; BerGenBio: Research Funding; Blueprint Medicine: Research Funding; Boehringer Ingelheim: Research Funding; Celgene: Research Funding; Daiichi Sankyo: Research Funding; Eisai: Research Funding; Gilead Sciences: Research Funding; Gylcotope GmbH: Research Funding; GlaxoSmithKline: Research Funding; Inside: Research Funding; IO Biotech: Research Funding; Isofol Medical: Research Funding; Janssen-Cilag: Research Funding; Karyopharm Therapeutics: Research Funding; Lilly: Research Funding; Millenium: Research Funding; MSD: Research Funding; Merck KGaA: Honoraria; Bayer: Honoraria, Research Funding; Roche: Honoraria, Research Funding; Merck Sharp Dohme: Consultancy, Honoraria; AstraZeneca: Honoraria, Research Funding; Lilly/ImClone: Consultancy; Nektar: Research Funding; Rafael Pharmaceuticals: Research Funding; Springworks Therapeutics: Research Funding; Taiho Pharmaceutical: Research Funding; Pfizer: Other. Fiedler: Celgene: Consultancy; Servier: Consultancy, Other: support for meeting attendance; Abbvie: Consultancy, Honoraria; Morphosys: Consultancy; Pfizer: Consultancy, Research Funding; Daiichi Sankyo: Consultancy, Other: support for meeting attendance; Jazz Pharmaceuticals: Consultancy, Other: support for meeting attendance; Stemline: Consultancy; Novartis: Consultancy; ARIAD/Incyte: Consultancy; Amgen: Consultancy, Other: support for meeting attendance, Patents & Royalties, Research Funding.

scholarly journals Combination of BCR-ABL1 Tyrosine Kinase Inhibitors with a Small Molecule LIM kinase1/2 Inhibitor in BCR-ABL1 Positive Acute Lymphoblastic Leukemia (ALL)

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2705-2705
Author(s):  
Thorsten Braun ◽  
Jeannig Berrou ◽  
Renaud Prudent ◽  
Hanane Djamai ◽  
Melanie Dupont ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Tyrosine Kinase ◽  
Cell Lines ◽  
Small Molecule ◽  
Research Funding ◽  
Kinase Inhibitors ◽  
Healthy Donors ◽  
Cd34 Cells

Abstract Introduction: LIM kinases 1 and 2 (LIMK1/2) are downstream effectors at the crossroads of different signaling pathways implicated in the dynamics of the cytoskeleton via phosphorylation of cofilin family proteins, degradation of the matrix by phosphorylating MT1-MMP and control of the activity of Aurora kinase A. Recently, the oncogenic role of Rho kinases (ROCK) was identified to be constitutively activated by BCR-ABL1, FLT3-ITD and KIT in hematologic malignancies via PI3 kinase and Rho GTPase mediated phosphorylation. Upon activation, ROCK phosphorylates LIMK1/2 leading to inactivation of cofilin by its phosphorylation and polymerization of actin and microtubules and possibly to other biological effects mediated by LIMK1/2, not yet fully understood. Here, we demonstrate synergy of a LIMK1/2 inhibitor with BCR-ABL1 tyrosine kinase inhibitors (TKI) in vitro and in vivo in different models for BCR-ABL1 driven ALL. Materials and Methods: Expression of LIMK1/2 was determined by RT-qPCR and WB in cell lines. Phosphorylation of cofilin was detected by WB. A small molecule inhibitor of LIMK1/2 was tested alone and in combination with imatinib, dasatinib, nilotinib and ponatinib in BCR-ABL1 positive ALL cell lines TOM-1 and BV-173. Cell viability and IC50 was assessed by MTS assays after exposure to LIMK1/2 inhibitor for 72h. In combination experiments, compounds were added simultaneously and relative cell numbers were determined at 72h with MTS assays and combination index (CI) values were calculated according to the Chou-Talalay model. Cell-cycle distribution was determined by cytofluorometric analysis detecting nuclear propidium iodide (PI) DNA intercalation. Induction of apoptosis was evaluated by annexin-V exposure and PI incorporation at 72h with increasing doses of LIMK1/2 inhibitor. Peripheral blood (PB) nucleated cells from apharesis products of healthy donors obtained after informed consent according to Helsinki declaration were incubated with or without LIMK1/2 inhibitor for 72h, and then enriched for CD34+ cells by immuno-magnetic selection and seeded in triplicate in methylcellulose FCS and cytokines. In vivo experiments were performed in C57Bl/6 mice injected with BCR-ABL-induced B-ALL cells. These were obtained by transduction of CDKN2A-deficient B-cell progenitors with a retrovirus coding for BCR-ABL1 (P185) and GFP, followed by transplantation in sub-lethally-irradiated recipient C57Bl/6 mice. Mice were treated either with LIMK1/2 inhibitor, nilotinib or the combination of both and compared to untreated control mice. Results: Expression of the two isoforms LIMK1 and LIMK2 in TOM-1 and BV-173 cells could be detected by RT-qPCR and at the protein level by WB. IC50 after LIMK1/2 inhibitor exposure alone was 580nM in TOM-1 cells and 1000nM in BV-173 cells. All combination experiments with the LIMK1/2 inhibitor and imatinib, dasatinib, nilotinib and ponatinib yielded synergistic CI for treatment of both TOM-1 and BV-173 cell lines. Cell cycle arrest in the G1/S transition was detected and LIMK1/2 inhibition induced dose dependent apoptosis in TOM-1 and BV-173 cells up to 40% at doses <1000nM. Upon treatment with the LIMK1/2 inhibitor, decrease of LIMK1 protein expression could be detected by WB, while LIMK2 expression was left unaffected. In both cell lines, LIMK1/2 inhibitor exposure lead to activating downstream dephosphorylation of cofilin as expected. No significant toxicity of increasing doses of LIMK1/2 inhibitor after exposure of CD34+ cells from healthy donors could be detected. To test the in vivo activity of LIMK1/2 inhibition, C57Bl/6 mice were transplanted with CDKN2Ako/BCR-ABL1+ B-ALL cells. Leukemic mice were treated with LIMK1/2 inhibitor alone, nilotinib or combination of LIMK1/2 inhibitor and nilotinib compared to untreated mice. The combination of nilotinib and LIMK1/2 inhibitor significantly delayed the appearance of leukemic cells in PB as detected by GFP+ cells once weekly or at death if possible with mice considered having leukemia if >1% GFP+ cells were detected in PB. Furthermore, nilotinib+LIMK1/2 inhibitor prolonged significantly the survival of mice compared to either nilotinib (p=0.0006) or LIMK1/2 inhibitor alone and untreated mice (p<0.0001) (Figure 1). Conclusion: Combination of LIMK1/2 inhibitor with BCR-ABL targeting TKI is synergistic and has significant anti-leukemic activity in BCR-ABL1+ ALL in vitro and in vivo models. Disclosures Braun: CELLIPSE: Research Funding. Prudent:CELLIPSE: Employment. Paublant:CELLIPSE: Employment. Baruchel:Jazz Pharmaceuticals: Consultancy, Honoraria, Other: Travel, accommodations or expenses; Shire: Research Funding; Servier: Consultancy; Amgen: Consultancy; Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; Roche: Consultancy. Dombret:CELLIPSE: Research Funding.

Use of Medium Potency Statins Is Associated with Improved Outcomes after Frontline RCHOP in Patients with Diffuse Large B-Cell Lymphoma (DLBCL)

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 45-45
Author(s):  
Sushanth Gouni ◽  
Paolo Strati ◽  
Jason Westin ◽  
Loretta J. Nastoupil ◽  
Raphael E Steiner ◽  
...  
Keyword(s):  
Cell Lines ◽  
Research Funding ◽  
Response Assessment ◽  
Cholesterol Content ◽  
In Vivo Studies ◽  
High Cholesterol ◽  
Improved Outcomes ◽  
Significant Difference

Background: Pre-clinical studies show that statins may improve the efficacy of chemoimmunotherapy in patients with DLBCL, through interference with cell membrane-initiated signaling pathways. Clinical retrospective studies, however, yield conflicting data, due to heterogeneous properties of statins, including potency and hydrophilicity. Methods: This is a retrospective analysis of patients with previously untreated, advanced stage DLBCL, non-double hit, treated with frontline R-CHOP between 01/01/2000 and 09/01/2019 (data cut-off 04/15/2020) at MD Anderson Cancer Center, and for whom data regarding statin use at time of initiation of treatment were available. Lugano 2014 response criteria were applied retrospectively for response assessment. Cellular cholesterol levels were analyzed in 6 DLBCL cell lines using an Amplex red fluorometric assay. A doxorubicin (DXR)-resistant cell line was generated exposing SUDHL4 cells to escalating doses of DXR; a DXR-resistant DLBCL patient-derived xenograft (PDX) model was established through serial transplantation and exposure to DXR. Results: 271 patients were included in the analysis, 182 (67%) were older than 60 years, 134 (49%) were male, 212 (72%) had stage IV disease, and 217 (80%) had an IPI score &gt; 3; upon pathological review, 38 (36%) cases were non-GCB type, and 18 (28%) were double-expressors; 214 (79%) were able to complete all planned 6 cycles of RCHOP. Seventy-nine (29%) patients received statins at time of initiation of chemoimmunotherapy: 15 patients received low potency statin, 51 medium and 13 high; 18 patients received hydrophilic statins and 61 lipophilic. Patients receiving statins were significantly older as compared to patients who did not (p&lt;0.001); no other significant difference in baseline characteristics was observed when comparing the 2 groups. Overall, 265 out of 271 patients were evaluable for response, as 6 stopped treatment because of toxicity before first response assessment. Among these, ORR was 95% (252/265) and CR rate was 62% (165/265). ORR rate was identical in patients who were treated with statin and those who did not (95% both, p=1). After a median follow-up of 77 months (95% CI, 70-84 months), 119 patients progressed/died, median PFS was not reached and 6-year PFS was 57%. 6-year PFS rate according to statin intensity was: 48% (low), 72% (medium), 57% (high). PFS. 6-year PFS rate was 64% for hydrophilic and 72% for lipophilic statins. Patients treated with statins had a trend for longer PFS (p=0.06), significantly longer for patients receiving medium potency statins (p=0.04). No significant difference in PFS was observed when comparing patients treated with lipophilic statins to all others (not reached vs 84 months, p=0.22). To confirm these clinical data, in-vitro and in-vivo studies were performed. Six cell lines were tested: 4 with high cholesterol content (SUDHL4, HBL1, HT, and U2932; 5.0-8.0 µg/mg protein), and 2 with low cholesterol content (DOHH2 and OCI-LY19; 1.5-2.0 µg/mg protein); the latter showed the highest sensitivity to DXR-mediated killing. The combination of lovastatin and DXR (10nM) was tested in all 4 cell lines with high cholesterol content, resulting in more cell death than either treatment alone. Lovastatin (at the nanomolar range) resensitized DXR-resistant SUDHL4 cells to DXR. Finally, in a DXR-resistant PDX model, the combination of lovastatin and DXR resulted in delayed tumor growth as compared to chemotherapy alone. Conclusions: Use of medium potency statins is associated with improved outcomes after frontline RCHOP in patients with DLBCL. This was further confirmed in functional in-vitro and in-vivo studies. Future interventional studies, aimed at improving outcomes in these patients using this novel combination, are warranted. Disclosures Westin: Amgen: Consultancy; 47: Research Funding; Kite: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Morphosys: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Curis: Consultancy, Research Funding; Astra Zeneca: Consultancy, Research Funding. Nastoupil:Gamida Cell: Honoraria; Merck: Research Funding; TG Therapeutics: Honoraria, Research Funding; Karus Therapeutics: Research Funding; Janssen: Honoraria, Research Funding; LAM Therapeutics: Research Funding; Novartis: Honoraria, Research Funding; Bayer: Honoraria; Celgene: Honoraria, Research Funding; Genentech, Inc.: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Gilead/KITE: Honoraria. Neelapu:Bristol-Myers Squibb: Other: personal fees, Research Funding; Merck: Other: personal fees, Research Funding; Kite, a Gilead Company: Other: personal fees, Research Funding; Pfizer: Other: personal fees; Celgene: Other: personal fees, Research Funding; Novartis: Other: personal fees; Karus Therapeutics: Research Funding; N/A: Other; Takeda Pharmaceuticals: Patents & Royalties; Acerta: Research Funding; Cellectis: Research Funding; Poseida: Research Funding; Precision Biosciences: Other: personal fees, Research Funding; Legend Biotech: Other; Adicet Bio: Other; Allogene Therapeutics: Other: personal fees, Research Funding; Cell Medica/Kuur: Other: personal fees; Calibr: Other; Incyte: Other: personal fees; Unum Therapeutics: Other, Research Funding. Landgraf:NCI/NIH: Research Funding. Vega:NCI: Research Funding.

Investigational Agent MLN2238/MLN9708, a Specific, Orally Available, Small Molecule Proteasome Inhibitor, Shows Promising In Vitro Activity Against Multiple Myeloma Cell Lines

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3014-3014
Author(s):  
Giada Bianchi ◽  
Vijay G. Ramakrishnan ◽  
Teresa Kimlinger ◽  
Jessica Haug ◽  
S. Vincent Rajkumar ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Er Stress ◽  
Cell Lines ◽  
Small Molecule ◽  
Proteasome Inhibitor ◽  
Research Funding ◽  
Proteasome Inhibitors ◽  
Biologically Active ◽  
Investigational Agent

Abstract Abstract 3014 Background: Proteasome inhibitors have proven particularly effective in treatment of multiple myeloma, the second most frequent hematologic malignancy in the western world. Bortezomib, the first in class proteasome inhibitor in clinical use, was first approved in 2003 via fast FDA track, given the remarkable activity shown during phase II clinical trials. Nevertheless, more than 50% of multiple myeloma patients did not respond to single agent bortezomib when administered as second line agent. Moreover, bortezomib is only available for intravenous administration, representing a cumbersome therapy for patients, and its use is limited by significant toxicities (especially peripheral neuropathy). MLN9708 (Millennium Pharmaceuticals, Inc.), an investigational orally available, small molecule, is a potent, specific and reversible inhibitor of the 20S proteasome. It is currently under clinical investigation for the treatment of hematologic and non-hematologic malignancies. Upon exposure to aqueous solutions or plasma, MLN9708 rapidly hydrolyzes to MLN2238, the biologically active form, and MLN2238 was used for all of the preclinical studies reported here. In vitro biochemistry studies have shown that MLN2238 has a faster dissociation rate from the proteasome compared to bortezomib, and in vivo studies of MLN2238 have shown antitumor activity in a broader range of tumor xenografts when compared to bortezomib. Given these encouraging preclinical results, we set to investigate the anti-myeloma activity of MLN2238 in vitro. Results: MLN2238 proved to have anti-proliferative and pro-apoptotic activity against a broad range of MM cell lines with EC50 at 24 hours ranging between 10 and 50 nM, even in relatively resistant MM cell lines (OPM2, DOX6, RPMI, etc.). In MM.1S cells, induction of apoptosis was time and dose dependent and related to activation of both caspase 8 and 9. When compared to MM.1S treated for 24 hours with EC50 dose of bortezomib, treatment with EC50 dose of MLN2238 resulted in the same extent of caspases cleavage occurring at an earlier time point (8-12 hours), possibly suggesting more rapid onset and/or irreversibility of apoptosis in cells treated with MLN2238. Treatment with MLN2238 was associated with early, but persistent induction of endoplasmic reticulum (ER) stress with BiP being induced 2–4 hours after treatment with EC50 dose and gradually increasing over time. While bortezomib has been associated with early induction and late decrease in proteins involved in ER stress, MLN2238 appears to induce a persistent rise in these factors, suggesting either more sustained proteasome blockade with stabilization of proteasome substrates or de-novo induction of unfolded protein response (UPR) genes. MLN2238 also proved effective in reducing phosphorylation of ERK1-2 with no overall alteration in the total ERK level, thus accounting for the observed reduction in proliferation upon treatment. Preliminary data indicate potential for additive and synergistic combination with widely used drugs, including doxorubicin and dexamethasone. Conclusion: While further clinical data are needed to establish the effectiveness of MLN2238 in the treatment of multiple myeloma, these preliminary nonclinical data, together with the favorable biochemical and pharmacokinetic properties, including oral bioavailability, make the investigational agent MLN9708 an appealing candidate for treatment of multiple myeloma. Further in vitro data could help establish whether a difference in the apoptotic mechanisms exist between MLN2238 and other proteasome inhibitors, primarily bortezomib, and could also help inform combination treatment approaches aimed at increasing effectiveness, overcoming bortezomib resistance and decreasing toxicity. Disclosures: Kumar: Celgene: Consultancy, Research Funding; Millennium: Research Funding; Merck: Consultancy, Research Funding; Novartis: Research Funding; Genzyme: Consultancy, Research Funding; Cephalon: Research Funding.

Recombinant Human Erythropoietin (rHuEPO) In Combination with Chemotherapy Increases Breast Cancer Metastasis In Pre-Clinical Mouse Models

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3345-3345
Author(s):  
Anargyros Xenocostas ◽  
Benjamin D Hedley ◽  
Jenny E Chu ◽  
D. George Ormond ◽  
Michel Beausoleil ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cell Lines ◽  
Research Funding ◽  
Breast Cancer Cells ◽  
Cancer Metastasis ◽  
Breast Cancer Metastasis ◽  
In Vivo Studies ◽  
Metastatic Process

Abstract Abstract 3345 Background: Erythropoietin (EPO) is a key regulator of erythropoiesis, and has been shown to stimulate growth, maintain viability, and promote differentiation of red blood cell precursors. The EPO receptor (EPO-R) is expressed by erythroid cells and by several non-hematopoietic cell types including various neoplastic cells. Erythropoiesis-stimulating agents (ESAs) are used clinically for the treatment of chemotherapy-induced anemia. The results of some recent randomized clinical trials have reported an increased incidence in adverse events and reduced survival in ESA-treated metastatic breast cancer patients receiving chemotherapy, potentially related to EPO-induced cancer progression. These results have raised concerns over ESA treatment in metastatic cancer patients. However, very little pre-clinical data is available regarding the impact of EPO on breast cancer metastasis. The goal of the current study was therefore to determine if EPO can influence the malignant behavior of breast cancer cells and/or influence the metastatic process. Methods: MDA-MB-468, MDA-MB-231, MDA-MB-435, and 4T-1 breast cancer cell lines were treated with recombinant human EPO (rHuEPO; 10 U/ml) or control media and screened for EPO-R mRNA expression levels by RT-PCR, and for EPO-R protein expression by Western blot and flow cytometry. MDA-MB-231 (231) and MDA-MB-435 (435) cell lines were used for functional assays in vitro and in vivo. Untreated or rHuEPO treated cells were grown in 2D and 3D in vitro systems (standard tissue culture plates and 0.6% soft agar, respectively) to determine if rHuEPO influenced growth. In vitro cell survival was also assessed in response to treatment with rHuEPO in the presence or absence of paclitaxel chemotherapy (10mg/ml), radiation (10G), or hypoxic conditions (1% O2). Following mammary fat pad injection, in vivo effects of rHuEPO (300U/kg) alone or in combination with paclitaxel treatment (10mg/kg) were assessed in mouse models of tumorigenicity and spontaneous metastasis. Results: Expression analysis of EPO-R mRNA and protein revealed a large variation in levels across different cell lines. The majority of cell lines did not express cell surface EPO-R by flow cytometry, although two cell lines (231 and 435) did show weak expression of EPO-R mRNA, with only the 231 cell line showing EPO-R expression by Western blot. In vitro, a small protective effect from rHuEPO on radiation-treated 435 cells was seen (p<0.05); however, rHuEPO treatment alone or combined with chemotherapy or hypoxia did not cause a significant increase in cell survival relative to untreated controls cells. In contrast, in vivo studies demonstrated that rHuEPO increased the incidence and burden of lung metastases in immunocompromised mice injected with 231 or 435 cells and treated with paclitaxel relative to mice treated with paclitaxel alone (p<0.05). Conclusions: The lack of an in vitro effect of rHuEPO highlights the importance of in vivo studies to delineate the effects of EPO on the metastatic process. Our novel findings demonstrate that rHuEPO can reduce the efficacy of chemotherapy in the metastatic setting in vivo, and in some cases enhance the inherent metastatic growth potential of human breast cancer cells. This work was supported by funding from the London Regional Cancer Program and Janssen Ortho Canada Disclosures: Xenocostas: Janssen Ortho: Consultancy, Honoraria, Research Funding. Allan:Janssen Ortho: 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.

SGI-110, a Novel Hypomethylating Agent, Induces the WNT Inhibitor Secreted Frizzled Related Protein-2 (SFRP2), and Down Regulates β-Catenin in Acute Myeloid Leukemia (AML) Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1290-1290
Author(s):  
Michelle Golding ◽  
Pragya Srivastava ◽  
Golda Collamat ◽  
Smitha R James ◽  
Adam R. Karpf ◽  
...  
Keyword(s):  
Western Blot ◽  
Cell Lines ◽  
Research Funding ◽  
Nuclear Translocation ◽  
Similarity Score ◽  
Cellular Distribution ◽  
U937 Cells ◽  
Fold Reduction ◽  
Wnt Inhibitor

Abstract Abstract 1290 Introduction: SGI-110 (Astex Pharmaceuticals, Inc.) is a dinucleotide hypomethylating agent whose active metabolite is decitabine (DAC). This drug demonstrates superior pharmacokinetics relative to the parent drug as a result of resistance to modification by cytidine deaminase, and is being investigated in myeloid malignancy in the phase I/II setting. We and others have demonstrated that WNT inhibitory genes including SFRP2 are epigenetically silenced in AML and that exposure to DNA methyltransferase inhibitors such as 5-Azacitidine (AZA) and DAC can re-express these genes and down-regulate β-catenin signaling in AML cell lines. We hypothesized that treatment with SGI-110 would have a similar effect upon the epigenetically silenced WNT inhibitor SFRP2 and further would down-regulate β-catenin signaling in AML cells in vitro. Methods: The AML cell lines HL60 and U937 were cultured in vitro using standard techniques and treated with phosphate buffered saline, 0.1, 1 or 5 μM SGI-110, 2μM AZA or 0.5μM DAC. Results presented are pooled data from a minimum of three biological replicates. Samples were harvested on day 5 and viable cells, DNA, RNA and protein obtained. β-catenin levels and cellular localization were quantified using imaging flow cytometry (ImageStream), DNA was extracted and bisulfite converted for analysis of gene specific and global DNA methylation by pyrosequencing (LINE-1, SFRP2), RNA was converted to cDNA for analysis by RT-PCR, and protein was obtained to confirm ImageStream results by Western blot. Nuclear translocation of β-catenin, indicative of its signaling activity, was assessed in individual cells by ImageStream using a similarity score: a log-transformed Pearson's correlation coefficient between the digitized images of immunostained β-catenin and a nuclear stain (DAPI). Shifts in the population (n=5,000) distributions of this similarity score were assessed by a resolution metric (Fishers discriminant ratio, Rd). Results: Treatment of AML cell lines with 5μM SGI-110 was toxic, and in line with previous experiments in AML cell lines, above the IC90. Treatment at the lowest dose of SGI-110 had minimal effects upon viability, methylation, and mRNA and protein expression in both cell lines tested. Treatment with SGI-110 at the 1μM dose resulted in reductions in LINE-1 methylation in HL60 cells by 21% (from 82% to 61%), compared to 8% with AZA (to 74%) and 20% with DAC (to 62%). In U937 cells, LINE-1 methylation decreased by 40% (from 67% to 27%) after SGI-110 treatment compared to a 25% reduction with AZA (to 42%) and a 30% reduction with DAC (to 36%). SFRP2 methylation in HL60 and U937 decreased from 86 and 88% at baseline to 66 and 60% with SGI-110 at the 1μM dose, compared to 68% with AZA and to 61% with DAC. Expression of SFRP2 mRNA was observed following treatment with 1μM SGI-110 and with DAC, but was limited following AZA treatment. ImageStream analysis of total cellular β-catenin in HL-60 and U937 cells demonstrated 2.4-fold and 1.2-fold reductions in total β-catenin following 1μM SGI-110 treatment. These results were similar to those seen with DAC (1.8-fold and 1.3-fold in HL-60 and U937 cells respectively). AZA treatment appeared to have a greater effect on total β-catenin in U937 cells (1.3-fold reduction) than in HL-60 cells (0.84-fold reduction). Western blot confirmed reductions in β-catenin protein. We also observed decreased nuclear translocation of β-catenin after treatment of HL-60 and U937 cells with 1 μM SGI-110 (Rd = −0.58 and −0.21 respectively; the negative sign indicates a change in cellular distribution from the nucleus to the cytoplasm). Changes were comparable to those observed with DAC (Rd = −0.75 and −0.26 in HL-60 and U937 cells respectively). AZA treatment of U937 cells resulted in a shift in cellular distribution (Rd = −0.20) similar to that for DAC and SGI-110 but had no effect on β-catenin distribution in HL-60 cells (Rd= 0.00). Conclusions: SGI-110 is a novel DNMT inhibitor which demonstrates robust effects on LINE-1 methylation, SFRP2 mRNA expression, and β-catenin level and localization consistent with epigenetically mediated re-expression of the WNT inhibitor SFRP2. Both upregulated β-catenin signaling and SFRP2 methylation have been demonstrated to correlate with inferior survival in patients with myeloid malignancies. Re-expression of epigenetically silenced WNT inhibitory genes such as SFRP2 may abrogate β-catenin signaling in AML cells. Disclosures: Karpf: Astex Pharmaceuticals: Research Funding. Griffiths:Celgene: Honoraria; Astex Pharmaceuticals: Research Funding.

Tumor Cell-Dependent Differences in Stroma-Induced Changes to Bortezomib Sensitivity.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2468-2468
Author(s):  
Eugen Dhimolea ◽  
Jana Jakubikova ◽  
Richard W.J. Groen ◽  
Jake E. Delmore ◽  
Hannah M. Jacobs ◽  
...  
Keyword(s):  
Tumor Cell ◽  
Tumor Cells ◽  
Cell Lines ◽  
Stromal Cells ◽  
Cell Response ◽  
Research Funding ◽  
Bioluminescence Imaging ◽  
Bortezomib Treatment ◽  
Rpmi 8226

Abstract Abstract 2468 In multiple myeloma (MM) and other hematologic malignancies, bone marrow stromal cells (BMSCs) confer resistance to diverse conventional or investigational therapeutics. During the last decade, data from many groups have concurred that the in vitro anti-MM activity of the proteasome inhibitor bortezomib is very similar in the presence and absence of BMSCs, including primary and immortalized BMSCs. These well-validated observations have supported the notion that novel, more effective, therapies for the treatment of MM should ideally be, similarly to bortezomib, capable of overcoming the protective effect of BMSCs. Interestingly, however, we have observed that primary CD138+ MM tumor cells isolated from patients with clinical refractoriness to bortezomib occasionally exhibit substantial in vitro response to clinically achievable concentrations of this drug. We therefore hypothesized that, under certain previously under-explored experimental settings, BMSCs may alter the threshold of MM cell response to bortezomib-induced apoptosis. To address this hypothesis in conditions that better simulate the clinical context, we conducted compartment-specific bioluminescence imaging (CS-BLI) assays to evaluate the effect of bortezomib on tumor cells co-cultured with BMSCs for different time periods prior to bortezomib administration. We observed that prolonged tumor-stromal co-culture (48–96hrs) prior to initiation of bortezomib treatment did not affect drug sensitivity for several MM cell lines (OPM2, H929, UM9, KMS11, KMS18 and RPMI-8226) tested. Prolonged co-culture of OPM1, RPMI-8226-Dox40, OCI-My5, KMS12BM and KMS18 cells prior to bortezomib treatment enhanced its activity. Importantly, extended co-culture of MM cell lines MM.1S and MM.1R with BMSCs prior to drug treatment induced significant attenuation of their response to bortezomib, as evidenced by 2–3 fold increase of IC50 values in several independent replicate experiments and a mean % area under the bortezomib dose response curve (AUC) of 5.82% vs 14.10% in the absence vs. presence of BMSCs, respectively (p=0.0079). Consistent with these in vitro results, heterotypic s.c. xenografts of Luc+ MM.1S cells mixed with Luc- BMSCs did not show statistically significant reduction in MM burden with bortezomib treatment (0.5 mg/kg s.c. twice weekly for 5 weeks) compared to vehicle-treated controls (p=0.1320), as quantified by bioluminescence imaging. In contrast, the same dose and schedule of bortezomib treatment significantly suppressed tumor burden, compared to vehicle-treated controls, of monotypic s.c. xenografts of Luc+ MM.1S cells in SCID mice (p=0.0022), as in prior experience. To evaluate the molecular mechanisms of cell non-autonomous decrease in MM cell response to bortezomib, we compared the transcriptional profiles of MM.1S cells in extended co-cultures with HS-5 BMSCs vs. MM.1S cells cultured in isolation. These studies identified a distinct transcriptional signature of stroma-induced transcripts, including several (e.g. PSMC3, ITGB7, FOS, ALDH1L2) for which transcript expression higher than the median levels for refractory MM patients correlated with shorter overall survival (p<0.02, log-rank tests) after treatment with bortezomib. These observations highlight the notion that tumor cell responses to a given agent in the presence of non-malignant stromal cells can exhibit substantial qualitative and quantitative variation, depending on the specific tumor cell type tested, as well as the particular stromal cell population and conditions of the co-culture. Our findings highlight the need to apply combinatorial high-throughput scalable platforms, such as CS-BLI, to evaluate the different permutations of interactions between tumor cells, non-malignant accessory cells of the microenvironment and administered therapeutics. This study also provides a comprehensive functional oncogenomic framework to identify prognostically relevant molecular mediators of stroma-induced resistance to therapy in MM. Disclosures: Groen: Genmab BV: Research Funding. McMilllin:Axios Biosciences: Equity Ownership. Mitsiades:Millennium Pharmaceuticals: Honoraria; Celgene: Honoraria; Novartis Pharmaceuticals: Honoraria; Bristol-Myers Squibb: Honoraria; Merck &Co.: Honoraria; Centocor: Honoraria; Arno Therapeutics: Honoraria; Amgen: Research Funding; AVEO Pharma: Research Funding; OSI: Research Funding; EMD Serono: Research Funding; Sunesis: Research Funding; Johnson & Johnson: Research Funding; PharmaMar: Licensing royalties Other; Axios Biosciences: Uncompensated Role as advisor, Uncompensated Role as advisor Other.

Genome Wide Studies in Multiple Myeloma Identify XPO1/CRM-1 As a Critical Target Validated Using the Selective Inhibitor of Nuclear Export (SINE) KPT-276

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 573-573
Author(s):  
Jessica Schmidt ◽  
Esteban Braggio ◽  
Marta Chesi ◽  
Jan Egan ◽  
Yuan Xiao Zhu ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
B Cell ◽  
Cell Lines ◽  
Nuclear Export ◽  
Research Funding ◽  
Phase 1 ◽  
A549 Cells ◽  
B Cell Lymphoma ◽  
Anticancer Activities

Abstract Abstract 573 Using high throughput RNA interference screening on 6,722 druggable genes we previously identified XPO1/CRM1 as one of the 50 most vulnerable targets in Multiple Myeloma (MM)1. XPO1 knockdown proved lethal in MM cell lines, but had no effect on human embryonic kidney (293) cells or lung cancer (A549) cells, showing that XPO1 is a specific myeloma vulnerability, and that myeloma cell survival is dependent upon XPO1 expression. XPO1 encodes the protein exportin 1, a nuclear transport protein that exports tumor suppressor proteins from the nucleus, where they are active, to the cytoplasm, where they become inactive. We next analyzed XPO1 in MM via gene expression profiling (GEP). XPO1 expression is up-regulated as the disease progresses: patients with active MM have a higher level of XPO1 compared to normal plasma cells (p<0.04) and to patients with monoclonal gammopathy of undetermined significance or smoldering MM (p<0.0001). The highest levels were in human MM cell lines. TC classification revealed highest levels in t(11;14) and lowest levels in t(4;14) disease. Selective inhibitors of nuclear export (SINE) compounds have recently been developed that irreversibly inhibit XPO1/CRM1 and its nuclear export function. One such inhibitor, KPT-276, decreased the viability of all 12 MM cell lines tested in vitro, as shown by MTT assay. After 72 hours of drug treatment, a median IC50 value of approximately 175 nM (range 30–1000 nM) was observed. No synergy with other commonly used anti-MM therapeutics was observed in vitro. In contrast, the drug had little effect in 8 solid tumor cell lines with the exception of the B cell lymphoma line Ramos. KPT-276 was also consistently active in inducing apoptosis against MM primary patient samples. Using an IC80 dose of KPT-276, drug-treated samples had a reduced population of cells in S phase (8%) compared to cells treated with DMSO (21%). Using the vkappa*myc transgenic MM model, KPT-276 reduced monoclonal spikes (by a mean of 56%) in all mice treated orally with 150 mg/kg dose three times per week for 4 weeks. Furthermore, KPT-276 significantly reduced tumor growth in a xenograft MM1.S mouse model. GEP was performed in the presence or absence of drug in two different MM cell lines. Two genes of probable relevance, cell division cycle 25 homolog A (CDC25A) and Bromodomain-containing protein 4 (BRD4), were dysregulated by SINE treatment. Both are involved in cell cycle control and have been linked to MYC. RT-PCR and western blotting confirm that MYC, CDC25A and BRD4 are down-regulated, as soon as six hours, after treatment with KPT-276. KPT-276 has shown marked anticancer activities against B cell malignancies in vitro and is active and tolerated in Phase I canine studies. KPT-330, a close analog of KPT-276, is currently in Phase 1 studies in human with advanced hematological and solid tumors. Disclosures: Schmidt: Karyopharm: Research Funding. McCauley:Karyopharm Therapeutics Inc: Employment. Shacham:Karyopharm Therapeutics: Employment. Kauffman:Karyopharm Therapeutics Inc: Employment. Stewart:Millenium: Consultancy, Honoraria, Research Funding; Onyx: Consultancy; Celgene: Consultancy.

Silencing The Sialyltransferase Gene ST3GAL6 Inhibits Adhesion and Migration Of Myeloma Cells In Vitro and Reduces The Homing and Proliferation Of Tumor Cells In Vivo

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 275-275
Author(s):  
Siobhan Glavey ◽  
Salomon Manier ◽  
Antonio Sacco ◽  
Michaela R Reagan ◽  
Yuji Mishima ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Research Funding ◽  
Advisory Board ◽  
Myeloma Cells ◽  
Sialyl Lewis ◽  
And Migration ◽  
Rpmi 8226

Abstract Background Glycosylation is a stepwise procedure of covalent attachment of oligosaccharide chains to proteins or lipids, and alterations in this process, especially increased sialylation, have been associated with malignant transformation and metastasis. The adhesion and trafficking of multiple myeloma (MM) cells is strongly influenced by glycosylation and multiple myeloma cells express a variety of adhesion molecules, including selectin ligands and integrins, which are typically dependent on glycosylation for their function. We have previously reported that the sialyltransferase ST3GAL6 is up-regulated in plasma cells from MM patients and that increased expression is associated with inferior overall survival (OS) in MM gene expression profiling (GEP) datasets. The functional significance of increased sialylation of MM cells has not previously been reported. Methods MM cell lines MM1s and RPMI-8226 were confirmed to have high expression levels of ST3GAL6 at the gene and protein level compared to healthy controls. Knockdown of ST3GAL6 was confirmed in MM cell lines RPMI-8226 and MM1s using lentiviral shRNAs targeting different regions in the ST3GAL6 mRNA. Specific ST3GAL6 knockdown was confirmed by reduced ST3GAL6 mRNA and protein expression in comparison to a scrambled control. In a calcein-AM fluorescence based adhesion assay we next evaluated the effects of ST3GAL6 knockdown on MM-cell adhesion to bone marrow stromal cells (BMSC’s) and fibronectin coated plates. Migration to 30nM SDF1-α was assessed using transwell plates comparing ST3GAL6 knockdown cells to scrambled controls. The commercially available sialyltransferase inhibitor 3Fax-Neu5Ac was used to pre-treat MM cells in vitro prior to assessment of apoptosis by flow cytometry. shST3GAL6 MM1s cells positive for green fluorescent protein and luciferin (GFP-Luc+) were injected into tail veins of SCID-Bg mice (5x106 cells, n=5/group) and mice were followed weekly using bioluminescent imaging (BLI) for tumor development. Bone marrow homing of tumor cells was assessed using in vivoconfocal imaging of the skull vasculature (n=3/group). Results Knockdown of ST3GAL6 in MM cell lines resulted in a 50% reduction in cell surface staining with the monoclonal antibody HECA-452. This indicated reduced expression of cutaneous lymphocyte associated antigen (CLA), a carbohydrate domain shared by sialyl Lewis X (sLex) and sialyl Lewis a (sLea) antigens, confirming suppression of ST3GAL6 activity. There was a significant reduction in the ability of knockdown cells to adhere to BMSC’s and fibronectin in-vitro compared to scrambled controls (P=0.016, 0.032 respectively). Migration ability of these cells in response to SDF1-α was also reduced (P=0.01). In vivo in a xenograft SCID-Bg mouse model shST3GAL6 cells demonstrated a reduced tumor burden as assessed by weekly BLI (P=0.017 at week 4). A consolidated map of the skull bone marrow niche in mice injected with shST3GAL6 MM1s GFP-Luc+ cells revealed a reduced homing ability of these cells in comparison to mice injected with scrambled control cells. Treatment of the MM cell lines MM1s and RPMI-8226 with a sialyltransferase inhibitor 3Fax-Neu5Ac resulted in almost complete elimination of cell surface sLex and/or sLea expression as determined by HECA-452 staining. Following pre-treatment with 3Fax-Neu5Ac, MM1S cells grown in co-culture with BMSC’s cells showed increased sensitivity to Bortezomib compared to cells treated with bortezomib alone. Conclusions shRNA knockdown of ST3GAL6 in MM cells significantly inhibits adhesion and migration in vitro with reduced homing and proliferation potential in vivo. In conjunction with the results of enzymatic inhibition this indicates that sialylation may play an important role in the malignant behavior of MM cells. Studies are ongoing to address the potential role of altered glycosylation in MM. Disclosures: Ghobrial: Onyx: Advisoryboard Other; BMS: Advisory board, Advisory board Other, Research Funding; Noxxon: Research Funding; Sanofi: Research Funding.

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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