Search

Purpose: Although R-CHOP–based immunochemotherapy cures significant proportions of patients with aggressive B-cell lymphoma, tumor cell susceptibility to chemotherapy varies, with mostly fatal outcome in cases of resistant disease. We and others have shown before that export of cytostatic drugs contributes to drug resistance. Now we provide a novel approach to overcome exosome-mediated drug resistance in aggressive B-cell lymphomas.Experimental Design: We used well-established centrifugation protocols to purify exosomes from DLBCL cell lines and detected anthracyclines using FACS and HPLC. We used shRNA knockdown of ABCA3 to determine ABCA3 dependence of chemotherapy susceptibility and monitored ABCA3 expression after indomethacin treatment using qPCR. Finally, we established an in vivo assay using a chorioallantoic membrane (CAM) assay to determine the synergy of anthracycline and indomethacin treatment.Results: We show increased efficacy of the anthracycline doxorubicin and the anthracenedione pixantrone by suppression of exosomal drug resistance with indomethacin. B-cell lymphoma cells in vitro efficiently extruded doxorubicin and pixantrone, in part compacted in exosomes. Exosomal biogenesis was critically dependent on the expression of the ATP-transporter A3 (ABCA3). Genetic or chemical depletion of ABCA3 augmented intracellular retention of both drugs and shifted the subcellular drug accumulation to prolonged nuclear retention. Indomethacin increased the cytostatic efficacy of both drugs against DLBCL cell lines in vitro and in vivo in a CAM assay.Conclusions: We propose pretreatment with indomethacin toward enhanced antitumor efficacy of anthracyclines and anthracenediones. Clin Cancer Res; 22(2); 395–404. ©2015 AACR.

Supp. Figure 1 (Detection of doxorubicin in the exosomes by HPLC and visualization of vesicular Transport); Supp. Figure 2 (DLBCL in ovo xenotransplantation model); Supp. Figure 3 (Increased cytotoxicity pixantrone and doxorubicin against DLBCL-ABC cell line OCI-Ly3 by pretreatment with indomethacin); Supp. Figure 4 (Celecoxib and omeprazole do not alter doxorubicin-susceptibility in SU-DHL-4 and OCI-Ly1 cells).

Supplementary figure legend

Tightly regulated activity of the transcription factor MYC is essential for orderly cell proliferation. Upon deregulation, MYC elicits and promotes cancer progression. Proteasomal degradation is an essential element of MYC regulation, initiated by phosphorylation at Serine62 (Ser62) of the MB1 region. Here, we found that Ser62 phosphorylation peaks in mitosis, but that a fraction of nonphosphorylated MYC binds to the microtubules of the mitotic spindle. Consequently, the microtubule-destabilizing drug vincristine decreases wild-type MYC stability, whereas phosphorylation-deficient MYC is more stable, contributing to vincristine resistance and induction of polyploidy. PI3K inhibition attenuates postmitotic MYC formation and augments the cytotoxic effect of vincristine. Implications: The spindle's function as a docking site for MYC during mitosis may constitute a window of specific vulnerability to be exploited for cancer treatment.

Purpose: Although R-CHOP–based immunochemotherapy cures significant proportions of patients with aggressive B-cell lymphoma, tumor cell susceptibility to chemotherapy varies, with mostly fatal outcome in cases of resistant disease. We and others have shown before that export of cytostatic drugs contributes to drug resistance. Now we provide a novel approach to overcome exosome-mediated drug resistance in aggressive B-cell lymphomas. Experimental Design: We used well-established centrifugation protocols to purify exosomes from DLBCL cell lines and detected anthracyclines using FACS and HPLC. We used shRNA knockdown of ABCA3 to determine ABCA3 dependence of chemotherapy susceptibility and monitored ABCA3 expression after indomethacin treatment using qPCR. Finally, we established an in vivo assay using a chorioallantoic membrane (CAM) assay to determine the synergy of anthracycline and indomethacin treatment. Results: We show increased efficacy of the anthracycline doxorubicin and the anthracenedione pixantrone by suppression of exosomal drug resistance with indomethacin. B-cell lymphoma cells in vitro efficiently extruded doxorubicin and pixantrone, in part compacted in exosomes. Exosomal biogenesis was critically dependent on the expression of the ATP-transporter A3 (ABCA3). Genetic or chemical depletion of ABCA3 augmented intracellular retention of both drugs and shifted the subcellular drug accumulation to prolonged nuclear retention. Indomethacin increased the cytostatic efficacy of both drugs against DLBCL cell lines in vitro and in vivo in a CAM assay. Conclusions: We propose pretreatment with indomethacin toward enhanced antitumor efficacy of anthracyclines and anthracenediones. Clin Cancer Res; 22(2); 395–404. ©2015 AACR.

Transcription factor NF-κB and its activating kinase IKKβ are associated with inflammation and are believed to be critical for innate immunity. Despite the likelihood of immune suppression, pharmacological blockade of IKKβ-NF-κB has been considered as a therapeutic strategy. However, we found neutrophilia in mice with inducible deletion of IKKβ (Ikkβ(Δ) mice). These mice had hyperproliferative granulocyte-macrophage progenitors and pregranulocytes and a prolonged lifespan of mature neutrophils that correlated with the induction of genes encoding prosurvival molecules. Deletion of interleukin 1 receptor 1 (IL-1R1) in Ikkβ(Δ) mice normalized blood cellularity and prevented neutrophil-driven inflammation. However, Ikkβ(Δ)Il1r1(-/-) mice, unlike Ikkβ(Δ) mice, were highly susceptible to bacterial infection, which indicated that signaling via IKKβ-NF-κB or IL-1R1 can maintain antimicrobial defenses in each other's absence, whereas inactivation of both pathways severely compromises innate immunity.

Abstract 3826 Poster Board III-762 Introduction The majority of patients with MDS depend on regular blood cell (RBC) transfusions during the course of their disease. Patients with lower-risk MDS are at particularly high risk of developing iron overload because of their longer median survival. Transfusional iron overload is known to be associated with increased morbidity mainly due to cardiac and/or hepatic damage. As a result an excess mortality rate in polytransfused pts. has been demonstrated. A negative prognostic impact of transfusion need is a proven independent marker for a bad prognosis. Jensen et al. (1996) demonstrated that an adequate chelation therapy could improve the transfusion need of pts. with MDS significantly (Br J Haematol 1996, 94, 288-299). This observation was supported by recent findings of another group (Messa, Acta Haematol, 2008, 120, 70-4) with improvement of transfusion need under adequate chelation therapy. Thus iron overload might not only be harmful to hepatocytes and cardiomyocytes but also to bone marrow progenitor cells. Their function is intrinsically impaired by MDS itself and might be further affected by a “second hit” in the form of toxic iron overload which might additionally impair their colony forming capacity. Patients and methods We performed colony assays from the peripheral blood from 52 pts. with MDS (RA/RARS: n=18, RCMD/RS: 12, RAEB-I/II: 13, 5q-syndrome: 3, MDS-U: 2, CMML: 1, and others: 2; age: 39 – 86 yrs. (median: 68 yrs.); cytogenetics: normal: 26, 5q-: 6, -7/7q-: 2, complex: 4, others: 4) with (serum ferritin ≥250 μg/L, range: 273 – 6267 μg/L, median: 664 μg/L) and without iron overload (range: 11 – 213 μg/L). Only pts. without hepatic and/or active infectious diseases, without chemotherapy/epigenetic therapy during the last 6 months and without cytokine and/or corticoid therapy during the last 3 months before performance of colony assays were considered. BFU-E and CFU-GM were analysed by the same person (U.S.) after 12 – 16 days in cultures from peripheral blood, performed as described (Leuk Res, 2001; 25(11):955-9) in 14 (BFU-E)/ 12 (CFU-GM) pts. with normal ferritin-values (normal range: 20-250 μg/L) in comparison to 38/32 pts. with ferritin values surmounting 250 μg/L. Pts. with diffuse growth or cluster formation (leukemic growth) were excluded. Statistical evaluation was performed with SAS 9.1 software using Wilcoxon-Mann-Whitney tests. The results were regarded as significant if the p-value was under 5%. Both patient subgroups were balanced according to cytogenetics, age and MDS WHO-subtype. Results In the patients subgroup with normal ferritin (n=14) the numbers of BFU-E ranged between 0 and 76 (std.dev. 19.63) with a median of 3.5 and a mean of 10.7, the numbers of CFU-GM ranged between 0.5 and 38.5 (std.dev. 13.23), with a median of 6.75 and a mean of 13.2. In the patients with elevated serum ferritin (n=38) the numbers of BFU-E ranged between 0 and 250 (std.dev.40.47) with a median of 0.5 and a mean of 8.86, the numbers of CFU-GM ranged between 0 and 120 (std.dev. 29.62) with a median of 3.0 and a mean of 18.94). Statistical comparison of the numbers of BFU-E and CFU-GM between patients with normal and elevated serum ferritin yielded a highly significant difference (p=0.001348) for BFU-E and no difference for CFU-GM (p=0.570296). Conclusions Our data provide further evidence that in MDS iron overload significantly impairs bone marrow function by suppression of the burst forming activity of erythroid progenitors. If this iron is removed by adequate chelation burst forming activity might be partially restored. Myeloid progenitors do not seem to be affected by iron overload. Perspective To address the question whether chelation therapy could improve erythropoiesis we performed 4 colony assays at minimum as follow up in 32pts the analysis of which is under way. In this group 8 pts. showed a normal ferritin while 26 pts. had an elevated ferritin (> 250 μg/L). Of these, pts. 10 were treated with chelation therapy. Furthermore, 9 pts. were monitored by magnetic resonance imaging (MRT). The results of these examinations will be related to the other parameters evaluated in this study and presented in detail. Disclosures: Haase: Novartis Oncology, Germany: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

In patients with myelodysplastic syndromes (MDS) iron overload caused by long-term red blood cell transfusions is an important factor for comorbidity especially in low-risk MDS. In this report we present the results of a comparative study based on colony formation assays of hematopoietic cells in MDS patients with and without iron overload. We demonstrate that iron overload suppresses the proliferation of erythroid progenitors cells (BFU-E), while the myeloid compartment (CFU-GM) was not found to be affected. Even patients with slightly elevated ferritin values show an impaired proliferation capacity in comparison to patients with normal ferritin levels. Furthermore, we show that this negative impact is reversible by sufficient iron chelation therapy. peerReviewed