Panchabhai, Sonali

In multiple myeloma (MM) the interaction of plasma cells, bone marrow stromal cells and tumor associated macrophages (TAMs) plays a significant role in conferring resistance to therapy and in the maintenance of residual disease. Folate receptors (FR) are specifically expressed on metabolically active malignant cells and TAMs and their expression in normal tissues and resting macrophages is limited. FR mediates folate uptake by receptor-mediated endocytosis. This qualifies the receptor to be exploited for drug delivery of folate conjugated cancer therapeutics. Our primary goal in this study is to evaluate the expression of functional FR in MM cells and TAMs with a view to exploit this for folate conjugated targeted therapy for MM. First we evaluated the presence of TAMs in paraffin embedded bone marrow slides of newly diagnosed MM patients with CD68 (pan-Macrophage marker) and CD163 antibodies (specific marker of TAMs) and found extensive infiltration of macrophages in bone marrow from MM patients. Next to evaluate expression of FR in MM cells, we employed an FR antibody and evaluated MM cell lines with immunoblot, flow cytometry and confocal microscopy. In a panel of ten MM cell lines, we found that out of the three FR isoforms (α, β and γ), FR beta (FR-β) is expressed by all of them. Next to test whether this expressed receptor is indeed functional, we incubated the cells with folate deficient media, added different concentrations of EC17 (folate conjugated to FITC, Endocyte Inc.) to the medium and looked for the uptake by flow cytometry after washing off the drug at different time points (after 10, 20, 40 and 60 min).We observed that the uptake begins in 10 min and is saturated at 1 hour with 100nM Folate-FITC. With confocal imaging, Folate-FITC was found in the cytoplasm of MM cells suggesting internalization of Folate-FITC and localization in the cytoplasm. In addition to myeloma cell lines, we also confirmed the uptake of Folate-FITC in CD138+ plasma cells of a newly diagnosed myeloma patient by flow cytometry. This strongly suggests that MM cells express functional FR-β. To test the specificity of this FR mediated uptake, we pre-incubated cells with 0.1mM folic acid in medium for 30 min and then added EC17. This maneuver blocked the activity mediated by FR and no uptake was observed , which proves that the Folate-FITC is internalized only through the FR. To evaluate the expression of FR in-vivo samples, we stained paraffin embedded bone marrow slides of newly diagnosed MM patients with FR-β antibody and TAM specific markers. We observe that FR is expressed on both MM cells as well as TAMs. To assess the endurance of cytotoxic effect of folate conjugated chemotherapeutic agents, we treated MM cell lines with folate conjugated vinka alkaloids and compared them to unconjugated drug and found no significant difference in their action suggesting conjugation with folate does not alter its efficacy. To assess potential toxicity of folate conjugated therapeutics, we obtained CD34+ cells and looked for the uptake of Folate-FITC with flow cytometry. We found no uptake and this is in line with previous reports suggesting that CD34 positive cells express nonfunctional FR. So we propose that FR qualify as potential targets for cancer treatment. Folate targeted therapy using folate-conjugated drugs which can selectively act against both MM cells and supporting TAMs has the potential of specific anti-MM tumoricidal activity. This therapeutic approach would broaden the use of drugs that could be conjugated with folate for MM therapy. Additionally assessment of TAMs in bone marrow sections of MM patients would add another feature for grading, classifying and prognosticating MM. Disclosures: Fonseca: Cylene: Research Funding; AMGEN: Consultancy; Millennium: Consultancy; Binding Site: Consultancy; Onyx: Consultancy, Research Funding; Lilly: Consultancy; BMS: Consultancy; Genzyme: Consultancy; Celgene: Consultancy; Medtronic: Consultancy; Otsuka: Consultancy; Prognostication of MM based on genetic categorization of the disease: Prognostication of MM based on genetic categorization of the disease, Prognostication of MM based on genetic categorization of the disease Patents & Royalties.

Background The precise molecular mechanism and targets of lenalidomide specific anti-Multiple Myeloma (MM) activity remain largely unknown, although, cereblon (CRBN) has been identified as a central mediator of activity with IRF-4 acting as a downstream molecule. Lenalidomide resistance in MM cells which, despite CRBN depletion, are able to restore their IRF-4 levels suggests the existence of alternative pathways. Methods To measure apoptosis and gene expression analysis 1x 106cells were incubated with 10μm to 50μm of lenalidomide for 24 to 96 hours before they were examined by annexin-PI and FACS analysis. Gene and protein expression were measured by RT-PCR, western blot, and immunohistochemistry. For lenalidomide followed bortezomib serial studies, cells were incubated with 10μm of lenalidomide for 48 hours and later treated with bortezomib for 24 to 48 hrs. CRBN and immunoglobulin light chain knock-down were performed by lentiviral mediated shRNA. Results We found lenalidomide induces Endoplasmic reticulum stress (ERS) via accumulation of intracellular immunoglobulin proteins, but only in the presence of CRBN in a panel of MM cell lines. This accumulation of intracellular proteins resulted in ERS as documented by western blot and RT-PCR in OPM2 and MMIS cell lines. We next utilized OPM2 cells knocked down for CRBN and control, non-target (NT), and treated with different doses of lenalidomide. After three days of treatment an ERS response was occurring in OPM2-NT cells but not in CRBN knockdown cells. Using Western blot we found decreased XBP-1u and increased XBP-1s, ERS marker proteins, in CRBN positive (NT) cells after 72 hours of lenalidomide treatment, but not in CRBN knock-down cells. Another ERS marker GRP78/BiP was also clearly accumulated after lenalidomide treatment in CRBN positive cells. Inactivation of the tumor suppressor p53 by degradation is a mechanism utilized by cells to adapt to ERS has been reported. We also found that p53 protein down regulated after lenalidomide treatment in CRBN positive OPM2 and MMIS cells. We have analyzed other isogenic cell lines MM1.S (lenalidomide-sensitive) and isogenic MM1.Sres (lenalidomide-resistant) HMCLs and found that lenalidomide induces ERS in MM1.S but not MM1.Sres cells. CRBN is important for lenalidomide to induce immunoglobulin protein accumulation and ERS was further proved by over-expressing CRBN in OCI-My5 MM cell line and subsequent lenalidomide treatment. To test the effect of light chain accumulation as ER stressors, we knocked down immunoglobulin light chain lambda in human MM cell line OPM2. Knock-down of lambda light chain in OPM2 cells resulted in lenalidomide resistance and it induced endoplasmic reticulum stress mediated anti-myeloma activity. Our preliminary data reveals that lenalidomide mediated progressive ERS can positively enhance bortezomib induced apoptosis in an in-vitro MM model. We pre-treated myeloma cell lines with lenalidomide and subsequently treated with bortezomib. MM cells pre-treated with lenalidomide for two days and there after treated with bortezomib clearly shown increased sensitivity to bortezomib induced apoptosis than non-pre-lenalidomide treated cells. Conclusion Here we show that lenalidomide causes accumulation of immunoglobulin light chains, inducing progressive ER stress, resulting in anti-myeloma activity and that effect depends on the presence of CRBN. Proteasome inhibitors such as bortezomib also sensitize MM cells to endoplasmic reticulum stress-mediated apoptosis. Our data demonstrated the synergistic effect of lenalidomide and proteasome inhibitors, likely due to the augmented protein stress. This data support the use of serial therapy utilizing pre-lenalidomide treatment followed by bortezomib or carfilzomib treatment. Disclosures: Fonseca: Medtronic: Consultancy; Otsuka: Consultancy; Celgene: Consultancy; Genzyme: Consultancy; BMS: Consultancy; Lilly: Consultancy; Onyx: Consultancy, Research Funding; Binding Site: Consultancy; Millennium: Consultancy; AMGEN: Consultancy; Cylene: Research Funding; Prognostication of MM based on genetic categorization of the disease: Prognostication of MM based on genetic categorization of the disease, Prognostication of MM based on genetic categorization of the disease Patents & Royalties.

Abstract Introduction Lenalidomide is an immunomodulatory drug (IMID) used to treat Multiple Myeloma (MM). Although a role for cereblon (CRBN)-mediated degradation of Ikaros proteins (IKZF1 and IKZF3) has been shown, the complete molecular and biochemical mechanisms responsible for lenalidomide-mediated anti-MM activity and/or resistance are undiscovered. Therefore, we aimed to analyze whether IMIDs (thalidomide, lenalidomide, and pomalidomide) are inducing oxidative stress in MM and what determines these drugs varying sensitivity and/or resistance. Methodology Amplex Red Assay has been performed to analyze IMIDs-mediated inhibition of H2O2 decomposition in both, in-vitro and in-vivo assays. Lentiviruses were prepared in 293T cells for CRBN, IgL-λ & IgL-k, and Bim knockdown experiment. Quantification of MM cellular anti-oxidative capacity for determining IMID sensitivity was standardized with H2O2-mediated oxidation of FADH2 and NAD(P)H. To measure apoptosis and gene expression analysis 106 cells were incubated with lenalidomide for 24 to 96 hours before they were examined by annexin-PI and FACS analysis. Gene and protein expression were measured by RT-PCR, western blot, and immunohistochemistry. Results We discovered that IMIDs inhibit peroxidase-mediated decomposition of H2O2 in both, in vitro horseradish peroxidase (HRP) assays and in human MM cell lines (HMCLs). Of the IMIDs analyzed, pomalidomide was the more potent inhibitor. H2O2 treatment effectively degraded IKZF1 and IKZF3 in HMCLs. To confirm the central role of CRBN in IKZF1 and IKZF3 degradation by H2O2-induced oxidative stress, we used CRBN knockdown OPM2 isogeneic cells and the CRBN-overexpressing OCIMY-5 cell line. We treated both sets of isogenic cell lines with lenalidomide and H2O2 for 3 hours, and we showed that H2O2 similarly mediates IKZF1 and IKZF3 degradation in a CRBN-dependent fashion. Next, we tested viability of CRBN present and absent cell lines with increasing concentrations of lenalidomide and H2O2 for 3 days. Lenalidomide-induced cytotoxicity was CRBN dependent, but H2O2 was not after 3 days, as shown by MTT assays. The capacity of MM cells to decompose H2O2 was measured via a biochemical test that quantitatively measured cellular anti-oxidative capacity. IMID sensitivity was well correlated with cellular anti-oxidative capacity, likely, cells more efficiently decompose H2O2was resistant and cells were not sensitive to IMID. This result shows that antioxidant capacity determines lenalidomide sensitivity among HMCLs with similar CRBN protein expression. We discovered that lenalidomide-mediated cytotoxicity in MM was attributable to oxidative damage of intracellular immunoglobulin proteins. By using several sets of isogenic cells lines with and without CRBN expression, we confirmed that lenalidomide treatment caused accumulation of IgL dimers only in CRBN-positive cells. Lenalidomide-induced IgL dimerization lead to decreased secretion and consequent intracellular accumulation of IgL, as evidenced by unchanged IgL mRNA expression, increased total intracellular IgL protein, and decreased secretion of IgL. After 72 hours of lenalidomide treatment we found decreased XBP-1u, increased XBP-1s, and over-expressed GRP78/BiP endoplasmic reticulum stress (ERS) marker proteins in CRBN positive cells but not in CRBN knock-down cells. We observed Bim requirement, especially BimEL, after lenalidomide treatment in CRBN-positive lenalidomide-sensitive cells. Our data reveals that lenalidomide-mediated; progressive ERS can positively enhance bortezomib-induced apoptosis in an in-vitro MM model. We pretreated MM cells with lenalidomide and then treated them with bortezomib. OPM2 cells pretreated with lenalidomide for 2 days clearly showed increased sensitivity to bortezomib-induced apoptosis compared with cells that were not pretreated. Conclusion IMIDs inhibit H2O2 decomposition. Ikaros protein degradation is a consequence of H2O2 mediated oxidative stress. Therefore, cells producing high H2O2 and with less antioxidative capacity are more sensitive to IMIDs. On the basis of this discovery, we would be able to predict which patients will benefit from IMIDs-mediated therapy and develop new drugs other than IMIDs that can inhibit intracellular H2O2 decomposition in MM. At present, CRBN may be required for IMIDs to effectively inhibit H2O2decomposition. Disclosures Chirackal: Mayo Clinic: Patents & Royalties: Filed a professional US patent for quantifying cellular anti-oxidative capacity. Fonseca:Mayo Clinic: Patents & Royalties: Filed a professional US patent for quantifying cellular anti-oxidative capacity.

Abstract Multiple myeloma (MM) is an age dependent second most common hematopoietic malignancy which remains incurable despite recent advances in therapies. Monoclonal gammopathy of undetermined significance (MGUS) is a common premalignant condition that precedes MM. Dysregulation and mutations of myriad of molecules is implicated in pathogenesis of MM. Cyclins (CCND) are almost universally dysregulated in MGUS and MM, while c-MYC overexpression and sometimes RAS mutations are associated with MGUS to MM progression. c-MYC, because of its strong association in this malignant transformation and it being a master regulatory factor is a logical therapeutic target. But, a therapeutic approach to target c-MYC has not been successful. So a strategy to target either upstream or downstream molecules in c-MYC pathway is worth considering. Ornithine decarboxylase (ODC) is one such downstream effector of c-MYC which regulates polyamine synthesis and thus regulates cell proliferation. ODC is also downstream of RAS which makes it common to two of the important oncogenes involved in MM. To know whether ODC plays a role in MM pathogenesis, we looked into its gene expression profile in the MM patients. In the Mayo cohort of 100 patients we found significant difference in ODC expression as disease progresses from MGUS to MM. We found significant survival difference in MM patients from this cohort which were divided by ODC expression and this survival difference was more pronounced in non-hyperdiploid group (median survival were for ODC < 1 - 66 mo vs for ODC > 1 - 29.5 mo, Figure 1A) which is a known poor prognostic group. When looked at ODC expression among different TC classes in MMRC dataset, we find ODC expression significantly higher in known high risk and poor prognostic groups 4p16 and MAF than other groups. These findings suggest higher ODC expression associated with poor survival. To further strengthen our observation, we analyzed TT3 group of Arkansas cohort and we observe prolonged event free survival (Figure 1B) and overall survival in patients with low ODC expression as compared to patients with high ODC expression (Figure 1C). After establishing poor prognostic role of ODC, we wanted to test it as a potential therapeutic target. For this purpose, we employed DFMO (Difluromethylornithine) which is the enzymatic irreversible inhibitor of ODC. We tested 15 different MM cell lines for proliferation with DFMO, majority of them respond to DFMO and IC 50 ranged from 28uM to 70 uM. DFMO generally halted cell cylce in G1S and had a cytostatic effect. We further tested efficacy of DFMO in combination with standard anti-myeloma agents lenalidomide, bortezomib, Vorinostat, melphalan and dexamethasone. We found these combinations to be synergistic except with melphalan where the combination was antagonistic. We therefore suggest that DFMO, which has a good toxicity profile can be advantageous in MM patients who are relatively old and many times cannot tolerate extensive chemotherapy for its toxicity. Moreover since it is synergistic in preclinical model with two main anti-myeloma agents lenalidomide and bortezomib, it may well be combined with both to decrease the amount of drug needed and hence toxicity. We think it will be especially beneficial to those patients who have high ODC levels. So we propose ODC to be a prognostic marker and therapeutic target in MM. Disclosures Chirackal: Mayo Clinic: Patents & Royalties: Filed a professional US patent for quantifying cellular anti-oxidative capacity. Fonseca:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Bayer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Onyx/Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Binding Site: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Millennium: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Applied Biosciences: Membership on an entity's Board of Directors or advisory committees.

Key Points IMiDs inhibit TrxR-mediated intracellular decomposition of H2O2 and caused oxidative stress in MM cells. MM cells with lower antioxidative capacity were more vulnerable to lenalidomide-induced H2O2 accumulation and its associated cytotoxicity.