ALBERT

Description: Abstract 2867 Background: Multiple myeloma (MM) is a highly resistant hematological neoplasia that remains an incurable disease. A leading drug in MM treatment is bortezomib, a selective proteasome inhibitor. Current treatment protocols have extended the overall survival of patients with MM, however, ultimately the disease becomes refractory to all forms of treatments and therefore drugs with new mechanisms of action are urgently needed. Experimental and clinical observations suggest that thyroid hormones (T3 and T4) modulate neoplastic cells and activate MAPK pathway through binding to αv β3 integrin, commonly overexpressed in cancer. Tetraiodothyroacetic acid (tetrac), a non-agonist T4 analog, selectively blocks T3 and T4 binding to αv β3 receptor site. MM cells interact with αv β3 for invasion/growth and thyroid diseases were associated with increased MM risk. We recently demonstrated the thyroid hormones- αv β3-MAPK axis in myeloma cells. In the current study we further show that T3 and T4 antagonize bortezomib action via MAPK activation and that in the presence of tetrac bortezomib action is significantly enhanced. Methods: Cell lines: MM cell lines (RPMI-8226, ARK, ARP-1, U266 and CAG) are cultured in RPMI 1640 supplemented with 10% heat-inactivated FBS/antibiotics. Before addition of T3 or T4, cells are grown for 48 hours without serum. Bone marrow (BM) aspirates were obtained from patients with MM treated at the Meir Medical Center. Signed institutional review board–approved written informed consent was obtained from all patients. Primary MM cells were separated on Ficoll gradient and were cultured in RPMI 1640. Reagents and chemicals: T3, T4, tetrac, MAPK inhibitor (U0126), autophagy inhibitor (3MA) and pan caspase inhibitor, Z-VAD. Cells were treated with T3 or T4 (1nM-100nM and 1μM) in the presence/absence of tetrac (100nM and 1μM) and/or bortezomib (25nM) and tested by several methods: Cell number. Cell proliferation assay: WST-1 (10% final concentration) is incubated at 37°C for 2 h and read using microELISA reader at 440nm. Cell cycle: Cells are harvested, fixed and stained with DNA propidium iodide (PI) (50 μg/ml) /RNAse A (10mg/ml) and analyzed for DNA content by FACS. Analysis of apoptosis/necrosis: Cells (105) are incubated with 5 μl Annexin V (FITC conjugated)/5 μl PI and analyzed by FACS. Expression of apoptotic genes (real-time PCR). Results were repeated 2–3 times in triplicates and were analyzed using unpaired students t test. Results and discussion: Results demonstrate that T3 and T4 at near physiological and supra physiological levels, increased myeloma cell viability by 15–50% and cell number by 30%-60%. This increased viability was blocked by U0126, indicating involvement of the MAPK pathway. In parallel a 20–25% reduction in cell death and of pro-apoptotic genes expression was documented following treatment with the hormones. Co-treatment of myeloma cell lines with T3 or T4 reduced bortezomib cytotoxicity and increased cell survival in a MAPK-dependent manner. Pretreatment of MM cell lines and primary cells from MM patients with tetrac, 48 hours before the addition of bortezomib, resulted in a synergistic cytotoxic effect. The effect of tetrac was blocked using a pan-caspase inhibitor (Z-VAD) but not by an autophagy inhibitor (3MA), suggesting apoptosis-related cell death. Conclusions: We present here novel data demonstrating that T3 and T4 may oppose bortezomib action via MAPK activation. Blocking the thyroid hormones- αv β3 axis using tetrac, promotes bortezomib cytotoxicity, suggesting this approach as a promising adjunct therapy in MM. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2964 Background: Basic/epidemiological/clinical data has established that thyroid hormones (T3/T4) may modulate neoplastic cells and T3/T4 deprivation removes this stimulus. Recently, growing body of epidemiological and clinical evidence suggests improved survival in individuals with primary/secondary hypothyroidism in a variety of tumor types, e.g., breast, lung, renal, prostate, head and neck, glioblastoma and more. On the other hand, hyperthyroidism was associated with an increased risk of lung and prostate cancers in a large-based population study as well as in pancreatic and ovarian cancers. Finally, enhanced response rates to chemo/radiation therapy in hypothyroidism patients have also been reported in preclinical/clinical studies. Taken together, these observations suggest a role for T3 and T4 in tumor growth and a protective effect of hypothyroidism in various cancer types. T3/T4 affects cell division/angiogenesis by activating MAPK/PI3K pathways through binding integrin αvβ3, an integrin over-expressed on many cancer cells. Thyroid binding site upon the integrin is at close proximity to its well known RGD recognition site and is blocked by tetrac, a natural analog of T4. Interestingly, in multiple myeloma (MM), a highly resistant hematological malignancy, the tumor cells interact with αvβ3 for their invasion/proliferation and thyroid diseases were associated with increased MM risk. As most MM patients still resist treatment, drugs with new mechanisms of action are urgently needed. We have recently demonstrated that, similar to results obtained from other cancer types, thyroid hormones induce myeloma cell viability and proliferation through MAPK activation. In the current study, we further studied the ability of thyroid hormones to induce MAPK activation in myeloma cells. We show that this MAPK induction is quick, specific and is initiated through thyroid hormones binding to the integrin αvβ3 receptor site and propose ways to block this potent action. Methods: Cell lines: MM cell lines, RPMI 8226 and CAG are cultured in RPMI 1640 supplemented with 10% heat-inactivated FBS/antibiotics. Reagents and chemicals: T3, T4, tetrac, RGD and RGE peptides, αvβ3 monoclonal antibodies (LM609), phosphorylated and total MAPK ERK1/2 antibodies. Cell proliferation assay: WST-1 (10% final concentration) is incubated at 37°C for 2 h and read using microELISA reader at 440nm. Flow cytometry: Cell cycle: Cells are harvested, fixed and stained with DNA propidium iodide (PI) (50 μ g/ml)/RNAse A (10mg/ml) and analyzed for DNA content by FACS. Analysis of apoptosis/necrosis: Cells (105) are incubated with 10 μ l Annexin V (FITC conjugated)/5 μ l PI and analyzed by FACS. Western blotting: Whole cell lysates were separated on 8–12% polyacrylamid gels and analyzed by western blot using above indicated antibodies. Results were repeated 2–4 times, in triplicates and were analyzed using unpaired students t test. Results and Discussion: Results demonstrate that physiological T3 and T4 concentrations (1nM and 100nM respectively), induce within minutes MAPK activation in myeloma cells, lasting up to 24 hours. This induction is completely blocked by a selective MAPK inhibitor, U0126. We further show that this thyroid-induced MAPK activation is initiated through αvβ3 integrin site, by using specific functional antibody to the integrin (LM609), as well as blocking the binding site using RGD but not RGE peptides. In addition, by using a T4 analog, tetrac, that selectively blocks thyroid hormones binding to the integrin site, we demonstrate a novel and specific way to block the induction of MAPK by T3 and T4. We present here indications that thyroid hormones induce a quick and potent MAPK activation in myeloma cells, similar to reports from other cancer types. This MAPK activation can be blocked using several inhibitors, including tetrac, previously demonstrated by us as a sensitizer of bortezomib action in myeloma. Conclusions: In order to surpass myeloma cancer treatment resistance, new mechanisms of action are urgently needed. Our unique approach, by inhibiting thyroid-induced MAPK pathway initiated at the αvβ3 integrin site, may potentially become a new promising chemosensitizing treatment in this disease. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2836 Poster Board II-812 Background: Multiple myeloma (MM) is a plasma cell neoplasia accounting for more than 10% of hematological malignancies. Since the disease was first described in England around 1850, MM has been very resistant to treatment with common relapses. It has a poor prognosis with a median survival of 3–5 years, despite all treatment approaches. In recent years, evidence has been provided that thyroid hormones (T3 and T4) may play a permissive role in various cancer cells including breast, brain, prostate and lung, enhancing tumor cell proliferation. Deprivation of these hormones decreases cancer cell proliferation and enhances cell death and response rates to chemotherapy and radiation therapy. It was recently discovered that T3 and T4 exert their proliferating actions through binding to aVb3 integrin, a common cell surface receptor, leading to mitogen-activated protein kinase (MAPK) activation and downstream intra cellular and nuclear events. Interestingly, aVb3 expression is increased during tumor progression and a spectrum of cancer cells, including MM, interact with this central integrin for their invasion, spreading and proliferation. In the current study, we hypothesized that that MM cells, similar to other cancer cells, are thyroid hormones sensitive and aimed to further investigate and characterize their effects on cell survival, proliferation and MAPK signaling. In addition, the additive/ supra additive effects of hypothyroid induction in MM cells on bortezomib's activity were evaluated. Methods: Cell lines: MM cell lines, RPMI 8226, U266, ARP1, ARK and CAG are cultured in RPMI 1640 supplemented with 10% heat-inactivated FBS/antibiotics. Reagents and chemicals: Bortezomib (Velcade) is obtained from the hospital pharmacy. T3, T4, tetrac RGD and RGE peptides (Sigma-Aldrich). PE conjugatedb3 monoclonal antibodies (LM609) and mouse IgG are from Chemicon International. phosphorylated MAPK ERK1/2, p38, JunK antibodies are from Cell Signaling (Danvers, MA). Alpha tubulin and PCNA antibodies are from Santa Cruz Biothecnology (Santa Cruz, CA, USA) WST-1 cell proliferation assay: WST-1 (10% final concentration) is incubated at 37°C for 2 h and read using microELISA reader at 440nm. Flow cytometry : Cell cycle: Cells are harvested, fixed and stained with DNA propidium iodide (PI) (50 μg/ml) /RNAse A (10mg/ml) and analyzed for DNA content by FACS. Analysis of apoptosis/necrosis: Cells (105) are incubated with 10 μl Annexin V (FITC conjugated)/5 μl PI and analyzed by FACS (Annexin+/PI-, early apoptosis; Annexin+/PI+, late apoptosis/necrosis). aVb3 in MM cells: Cells are harvested in RPMI 1640 and directly labeled with PE-aVb3 mAbs (10 mg/ml) and analyzed by FACS. Isotype-matched antibody, serves as negative control.Western blotting: Whole cell lysates were separated on 5-8% polyacrylamid gels and analyzed by western blot using antibodies for phosphorylated MAPK ERK1/2, p38, JunK and PCNA.Statistical analyses: Results were analyzed using unpaired students t test. Results: The sensitivity of myeloma cells to thyroid hormones was explored by addition of increasing concentrations of T3 and T4 to several myeloma cell lines. Results demonstrate that T3 and T4 significantly induced proliferation and cell number in these cells in accordance with PCNA protein elevation. This proliferating action was MAPK related, with phosphor ERK1/2, p38 and JunK elevated in a dose dependent manner. Mimicking hypothyroidism in the cells by using condition medium or T4 analog that block thyroid hormones binding to the integrin, tetrac, inhibited proliferation, increased apoptosis/necrosis and produced G2M arrest. Moreover, supra additive/additive “drug sparing” effects of tetrac-botezomib were observed with significant reduction in survival and increase in apoptosis. Discussion: We present here, for the first time in myeloma, indication that myeloma cells, similar tp reports from other cancer types, are thyroid hormones sensitive and that hypothyroidism induction inhibits cell proliferation and sensitizes response to bortezomib. Conclusions: As most MM patients still relapse, new drugs combinations are needed to overcome resistance. Our novel chemosensitizing approach may potentially demonstrate the importance of thyroid hormones status in this disease and may suggest a protective effect of sub clinical hypothyroidism in MM as a useful and unique adjunct for MM therapy. Disclosures: No relevant conflicts of interest to declare.