Targeting transcription regulation in cancer with a covalent CDK7 inhibitor.

Tumour oncogenes include transcription factors that co-opt the general transcriptional machinery to sustain the oncogenic state, but direct pharmacological inhibition of transcription factors has so far proven difficult. However, the transcriptional machinery contains various enzymatic cofactors that can be targeted for the development of new therapeutic candidates, including cyclin-dependent kinases (CDKs). Here we present the discovery and characterization of a covalent CDK7 inhibitor, THZ1, which has the unprecedented ability to target a remote cysteine residue located outside of the canonical kinase domain, providing an unanticipated means of achieving selectivity for CDK7. Cancer cell-line profiling indicates that a subset of cancer cell lines, including human T-cell acute lymphoblastic leukaemia (T-ALL), have exceptional sensitivity to THZ1. Genome-wide analysis in Jurkat T-ALL cells shows that THZ1 disproportionally affects transcription of RUNX1 and suggests that sensitivity to THZ1 may be due to vulnerability conferred by the RUNX1 super-enhancer and the key role of RUNX1 in the core transcriptional regulatory circuitry of these tumour cells. Pharmacological modulation of CDK7 kinase activity may thus provide an approach to identify and treat tumour types that are dependent on transcription for maintenance of the oncogenic state.

Compartment-Specific Bioluminescence Imaging platform for the high-throughput evaluation of antitumor immune function.

Conventional assays evaluating antitumor activity of immune effector cells have limitations that preclude their high-throughput application. We adapted the recently developed Compartment-Specific Bioluminescence Imaging (CS-BLI) technique to perform high-throughput quantification of innate antitumor activity and to show how pharmacologic agents (eg, lenalidomide, pomalidomide, bortezomib, and dexamethasone) and autologous BM stromal cells modulate that activity. CS-BLI-based screening allowed us to identify agents that enhance or inhibit innate antitumor cytotoxicity. Specifically, we identified compounds that stimulate immune effector cells against some tumor targets but suppressed their activity against other tumor cells. CS-BLI offers rapid, simplified, and specific evaluation of multiple conditions, including drug treatments and/or cocultures with stromal cells and highlights that immunomodulatory pharmacologic responses can be heterogeneous across different types of tumor cells. This study provides a framework to identify novel immunomodulatory agents and to prioritize compounds for clinical development on the basis of their effect on antitumor immunity.

Methyljasmonate displays in vitro and in vivo activity against multiple myeloma cells.

Jasmonates, plant stress hormones, have been demonstrated to be effective in killing various types of cancer cells. We therefore tested if methyljasmonate (MJ) has activity against multiple myeloma (MM) in vitro and in vivo. MM cell lines and primary MM tumour cells responded to MJ in vitro at concentrations that did not significantly affect normal haematopoietic cells, without stroma-mediated resistance. Brief MJ exposures of MM cells caused release of Hexokinase 2 (HK2) from mitochondria, rapid ATP depletion, perturbation of major intracellular signalling pathways, and ensuing mainly apoptotic cell death. Sensitivity to MJ correlated with lower cellular glucose consumption and lactate production, as well as lower intracellular protein levels of HK2, phosphorylated Voltage-dependent anion channel 2/3 (pVDAC2/3) and Aldo-keto reductase family 1 member C1 (AKR1C1), which represent potential biomarkers of responsiveness to MJ treatment, especially as AKR1C1 transcript levels also correlate with clinical outcome in bortezomib- or dexamethasone-treated MM patients. Interestingly, MJ synergized with bortezomib in vitro and prolonged survival of immunocompromised mice harbouring diffuse lesions of MM.1S cells compared to vehicle-treated mice (P = 0·0046). These studies indicate that jasmonates represent a new, promising strategy to treat MM.

Adenosine A2A receptor agonists and PDE inhibitors: a synergistic multitarget mechanism discovered through systematic combination screening in B-cell malignancies.

Using a combination high-throughput screening technology, multiple classes of drugs and targeted agents were identified that synergize with dexamethasone (Dex) in multiple myeloma (MM) cells. Performing combination screening with these enhancers, we discovered an unexpected synergistic interaction between adenosine receptor agonists and phosphodiesterase (PDE) inhibitors that displays substantial activity in a panel of MM and diffuse large B-cell lymphoma (DLBCL) cell lines and tumor cells from MM patients. We have used selective adenosine receptor agonists, antagonists, and PDE inhibitors as well as small interfering RNAs targeting specific molecular isoforms of these proteins to dissect the molecular mechanism of this synergy. The adenosine A2A receptor and PDE2, 3, 4, and 7 are important for activity. Drug combinations induce cyclic AMP (cAMP) accumulation and up-regulate PDE4B. We also observe rigorous mathematical synergy in 3-way combinations containing A2A agonists, PDE inhibitors, and Dex at multiple concentrations and ratios. Taken together, these data suggest that A2A agonist/PDE inhibitor combinations may be attractive as an adjunctive to clinical glucocorticoid containing regiments for patients with MM or DLBCL and confer benefit in both glucocorticoid-sensitive and -resistant populations.

Inhibition of tumor cells interacting with stromal cells by xanthones isolated from a Costa Rican Penicillium sp.

CR1642D, an endophytic isolate of Penicillium sp. collected from a Costa Rican rainforest, was identified through a high-throughput approach to identify natural products with enhanced antitumor activity in the context of tumor-stromal interactions. Bioassay-guided separation led to the identification of five xanthones (1-5) from CR1642D. The structures of the xanthone dimer penexanthone A (1) and monomer penexanthone B (2) were elucidated on the basis of spectroscopic analyses, including 2D NMR experiments. All of the compounds were tested against a panel of tumor cell lines in the presence and absence of bone marrow stromal cells. Compound 3 was the most active, with IC(50) values of 1-17 µM, and its activity was enhanced 2-fold against tumor cell line RPMI8226 in the presence of stromal cells (IC(50) 1.2 µM, but 2.4 µM without stromal cells).

Molecular and cellular effects of multi-targeted cyclin-dependent kinase inhibition in myeloma: biological and clinical implications.

Cell cycle regulators, such as cyclin-dependent kinases (CDKs), are appealing targets for multiple myeloma (MM) therapy given the increased proliferative rates of tumour cells in advanced versus early stages of MM. We hypothesized that a multi-targeted CDK inhibitor with a different spectrum of activity compared to existing CDK inhibitors could trigger distinct molecular sequelae with therapeutic implications for MM. We therefore studied the small molecule heterocyclic compound NVP-LCQ195/AT9311 (LCQ195), which inhibits CDK1, CDK2 and CDK5, as well as CDK3 and CDK9. LCQ195 induced cell cycle arrest and eventual apoptotic cell death of MM cells, even at sub-µmol/l concentrations, spared non-malignant cells, and overcame the protection conferred to MM cells by stroma or cytokines of the bone marrow milieu. In MM cells, LCQ195 triggered decreased amplitude of transcriptional signatures associated with oncogenesis, drug resistance and stem cell renewal, including signatures of activation of key transcription factors for MM cells e.g. myc, HIF-1α, IRF4. Bortezomib-treated MM patients whose tumours had high baseline expression of genes suppressed by LCQ195 had significantly shorter progression-free and overall survival than those with low levels of these transcripts in their MM cells. These observations provide insight into the biological relevance of multi-targeted CDK inhibition in MM.

Anti-tumor activity and signaling events triggered by the isothiocyanates, sulforaphane and phenethyl isothiocyanate, in multiple myeloma.

BACKGROUND: Isothiocyanates, a family of phytochemicals found in cruciferous vegetables, have cytotoxic effects against several types of tumor cells. Multiple myeloma is a fatal disease characterized by clonal proliferation of plasma cells in the bone marrow. The growing body of preclinical information on the anti-cancer activity of isothiocyanates led us to investigate their anti-myeloma properties. DESIGN AND METHODS: We evaluated the anti-myeloma activity of the isothiocyanates, sulforaphane and phenethyl isothiocyanate, on a panel of human myeloma cell lines as well as primary myeloma tumor cells. Cell viability, apoptosis, cell cycle alterations and cell proliferation were then analyzed in vitro and in a xenograft mouse model in vivo. The molecular sequelae of isothiocyanate treatment in multiple myeloma cells were evaluated by multiplex analyses using bead arrays and western blotting. RESULTS: We observed that sulforaphane and phenylethyl isothiocyanate have activity against myeloma cell lines and patients' myeloma cells both in vitro and in vivo using a myeloma xenograft mouse model. Isothiocyanates induced apoptotic death of myeloma cells; depletion of mitochondrial membrane potential; cleavage of PARP and caspases-3 and -9; as well as down-regulation of anti-apoptotic proteins including Mcl-1, X-IAP, c-IAP and survivin. Isothiocyanates induced G(2)/M cell cycle arrest accompanied by mitotic phosphorylation of histone H3. Multiplex analysis of phosphorylation of diverse components of signaling cascades revealed changes in MAPK activation; increased phosphorylation of c-jun and HSP27; as well as changes in the phosphorylation of Akt, and GSK3α/ß and p53. Isothiocyanates suppressed proliferation of myeloma cells alone and when co-cultured with HS-5 stromal cells. Sulforaphane and phenylethyl isothiocyanate enhanced the in vitro anti-myeloma activity of several conventional and novel therapies used in multiple myeloma. CONCLUSIONS: Our study shows that isothiocyanates have potent anti-myeloma activities and may enhance the activity of other anti-multiple myeloma agents. These results indicate that isothiocyanates may have therapeutic potential in multiple myeloma and provide the preclinical framework for future clinical studies of isothiocyanates in multiple myeloma.

Activation, immune polarization, and graft-versus-leukemia activity of donor T cells are regulated by specific subsets of donor bone marrow antigen-presenting cells in allogeneic hemopoietic stem cell transplantation.

We investigated the roles of specific subsets of donor APCs purified from bone marrow in donor T cell activation and graft-vs-leukemia (GvL) activity in murine models of hemopoietic stem cell transplantation. Lineage(-)CD11c(+) APC precursors were separated from donor bone marrow based on expression of CD11b. Transplanting lineage(-)CD11c(+)CD11b(-) APC (CD11b(-) APC) in combination with c-kit(+)Sca-1(+)lineage(-) hemopoietic stem cells (HSC) and congenic donor T cells led to increased donor CD4(+) and CD8(+) T cell proliferation and higher donor T cell chimerism than with transplanting grafts containing HSC, T cells, and lineage(-)CD11c(+)CD11b(+) APCs (CD11b(+) APC), or grafts containing only HSC and T cells. Transplanting CD11b(-) APCs induced Th1/type 1 cytotoxic T lymphocyte donor T cell immune polarization and enhanced GvL activity of donor T cells without increased graft-vs-host disease in both MHC- and minor histocompatibility Ag-mismatched murine hemopoietic stem cell transplantation models, whereas CD11b(+) APCs led to Th2/type 2 cytotoxic T lymphocyte donor T cell immune polarization. Donor CD11b(-) APCs were plasmacytoid dendritic cell progenitors (>90% CD317; PDCA-1(+)) and up-regulated CD80, CD86, and IL-12 during alloantigen presentation, whereas CD11b(+) APCs expressed Gr-1 and up-regulated expression of programmed death ligands-1 and 2 after activation. These results are the first to show that manipulation of the content of donor APCs in allogeneic HSC grafts can regulate donor T cell immunity and enhance GvL without increasing graft-vs-host disease activity.

Emerging treatments for multiple myeloma: beyond immunomodulatory drugs and bortezomib.

The successful clinical development of thalidomide, bortezomib, and lenalidomide not only transformed the therapeutic management of multiple myeloma (MM) but also catalyzed a renewed interest in the development of additional classes of novel agents for this disease. This review focuses on a series of new therapeutics that have shown promising preclinical results, as well as encouraging safety profiles and early evidence of anti-MM activity in clinical studies, either alone or in combination with other, conventional or novel, anti-MM treatments. These agents include second-generation proteasome inhibitors and immunomodulatory agents, as well as members of other therapeutic classes, such as histone deacetylase inhibitors (HDAC), heat shock protein 90 (Hsp90) inhibitors, and the alkylphospholipid Akt inhibitor perifosine.

In vitro anti-myeloma activity of the Aurora kinase inhibitor VE-465.

This study characterized the preclinical anti-myeloma activity of VE465, a low molecular weight pan-Aurora kinase inhibitor. After 96-h drug exposure, several multiple myeloma (MM) cell lines were more sensitive to VE465 compared to non-malignant cells. The anti-MM activity of VE465 was maintained in the presence of interleukin-6 and, interestingly, enhanced by co-culture with stromal cells. However, primary MM cells were less responsive than cell lines. Combinations with dexamethasone (Dex), doxorubicin (Doxo) and bortezomib showed no antagonism. Our study highlights the potential role of the tumour microenvironment in modulating the activity of this drug class.

Stromal-mediated protection of tyrosine kinase inhibitor-treated BCR-ABL-expressing leukemia cells.

Clinical studies of patients with chronic myeloid leukemia revealed that a common pattern of response is a dramatic fall in the circulating population of blast cells, with a minimal or delayed decrease in marrow blasts, suggesting a protective environment. These observations suggest that a greater understanding of the interaction of stromal cells with leukemic cells is essential. Here, we present an in vivo system for monitoring relative tumor accumulation in leukemic mice and residual disease in leukemic mice treated with a tyrosine kinase inhibitor and an in vitro system for identifying integral factors involved in stromal-mediated cytoprotection. Using the in vivo model, we observed high tumor burden/residual disease in tissues characterized as significant sources of hematopoiesis-promoting stroma, with bone marrow stroma most frequently showing the highest accumulation of leukemia in untreated and nilotinib-treated mice as well as partial protection of leukemic cells from the inhibitory effects of nilotinib. These studies, which showed a pattern of leukemia distribution consistent with what is observed in imatinib- and nilotinib-treated chronic myeloid leukemia patients, were followed by a more in-depth analysis of stroma-leukemia cell interactions that lead to protection of leukemia cells from nilotinib-induced cytotoxicity. For the latter, we used the human BCR-ABL-positive cell line, KU812F, and the human bone marrow stroma cell line, HS-5, to more closely approximate the bone marrow-associated cytoprotection observed in drug-treated leukemia patients. This in vitro system helped to elucidate stromal-secreted viability factors that may play a role in stromal-mediated cytoprotection of tyrosine kinase inhibitor-treated leukemia cells.

Targeting BRAFV600E in thyroid carcinoma: therapeutic implications.

B-Raf is an important mediator of cell proliferation and survival signals transduced via the Ras-Raf-MEK-ERK cascade. BRAF mutations have been detected in several tumors, including papillary thyroid carcinoma, but the precise role of B-Raf as a therapeutic target for thyroid carcinoma is still under investigation. We analyzed a panel of 93 specimens and 14 thyroid carcinoma cell lines for the presence of BRAF mutations and activation of the mitogen-activated protein/ERK kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway. We also compared the effect of a B-Raf small inhibitory RNA construct and the B-Raf kinase inhibitor AAL881 on both B-Raf wild-type and mutant thyroid carcinoma cell lines. We found a high prevalence of the T1799A (V600E) mutation in papillary and anaplastic carcinoma specimens and cell lines. There was no difference in patient age, B-Raf expression, Ki67 immunostaining, or clinical stage at presentation between wild-type and BRAF(V600E) specimens. Immunodetection of phosphorylated and total forms of MEK and ERK revealed no difference in their phosphorylation between wild-type and BRAF(V600E) patient specimens or cell lines. Furthermore, a small inhibitory RNA construct targeting the expression of both wild-type B-Raf and B-Raf(V600E) induced a comparable reduction of viability in both wild-type and BRAF(V600E) mutant cancer cells. Interestingly, AAL881 inhibited MEK and ERK phosphorylation and induced apoptosis preferentially in BRAF(V600E)-harboring cells than wild-type ones, possibly because of better inhibitory activity against B-Raf(V600E). We conclude that B-Raf is important for the pathophysiology of thyroid carcinomas irrespective of mutational status. Small molecule inhibitors that selectively target B-Raf(V600E) may provide clinical benefit for patients with thyroid cancer.

Potentiation of antileukemic therapies by Smac mimetic, LBW242: effects on mutant FLT3-expressing cells.

Members of the inhibitor of apoptosis protein (IAP) family play a role in mediating apoptosis. Studies suggest that these proteins may be a viable target in leukemia because they have been found to be variably expressed in acute leukemias and are associated with chemosensitivity, chemoresistance, disease progression, remission, and patient survival. Another promising therapeutic target, FLT3, is mutated in about one third of acute myelogenous leukemia (AML) patients; promising results have recently been achieved in clinical trials investigating the effects of the protein tyrosine kinase inhibitor PKC412 on AML patients harboring mutations in the FLT3 protein. Of growing concern, however, is the development of drug resistance resulting from the emergence of point mutations in targeted tyrosine kinases used for treatment of acute leukemia patients. One approach to overriding resistance is to combine structurally unrelated inhibitors and/or inhibitors of different signaling pathways. The proapoptotic IAP inhibitor, LBW242, was shown in proliferation studies done in vitro to enhance the killing of PKC412-sensitive and PKC412-resistant cell lines expressing mutant FLT3 when combined with either PKC412 or standard cytotoxic agents (doxorubicin and Ara-c). In addition, in an in vivo imaging assay using bioluminescence as a measure of tumor burden, a total of 12 male NCr-nude mice were treated for 10 days with p.o. administration of vehicle, LBW242 (50 mg/kg/day), PKC412 (40 mg/kg/day), or a combination of LBW242 and PKC412; the lowest tumor burden was observed in the drug combination group. Finally, the combination of LBW242 and PKC412 was sufficient to override stromal-mediated viability signaling conferring resistance to PKC412.

Bcl-2 overexpression in thyroid carcinoma cells increases sensitivity to Bcl-2 homology 3 domain inhibition.

CONTEXT: The Bcl-2 family of proteins regulates apoptosis in various models and may represent a promising therapeutic target in human malignancies. OBJECTIVE/METHODS: We evaluated the sensitivity of thyroid carcinoma cell lines (two papillary, one follicular, two anaplastic, three medullary) in vitro to BH3I-1 and BH3I-2', two cell-permeable inhibitors of the Bcl-2 homology (BH)-3 domain-mediated interaction between proapoptotic and antiapoptotic Bcl-2 family members. The thyroid carcinoma cell line FRO was stably transfected with cDNA for Bcl-2 or constitutively active Akt and evaluated for sensitivity to BH3-domain inhibition. RESULTS: BH3-domain inhibition disrupted the mitochondrial membrane potential in thyroid carcinoma cells, induced caspase-dependent apoptosis, and potently sensitized them to sublethal concentrations of doxorubicin and the proteasome inhibitor bortezomib (Velcade). Overexpression of constitutively active Akt suppressed BH3I-1-induced cell death. Bcl-2-overexpressing FRO cells were more resistant to conventional chemotherapeutic agents (such as doxorubicin) but significantly more sensitive to BH3I-1 than control cells and were found to overexpress caspase-9, caspase-8, Bmf, Bok, and Bik transcripts and express less A1, BRaf, and FLIP transcripts. CONCLUSIONS: Bcl-2 expression protects thyroid carcinomas against chemotherapy-induced apoptosis. Nevertheless, overexpression of Bcl-2 may result in "oncogene addiction" of the cancer cell, which can be exploited by using BH3-domain inhibitors alone or in combination with other agents, including conventional chemotherapeutics (such as doxorubicin) or novel targeted therapies (such as the proteasome inhibitor bortezomib), for the treatment of aggressive thyroid cancer, including the medullary and anaplastic types.

The proteasome inhibitor bortezomib induces apoptosis in human retinoblastoma cell lines in vitro.

PURPOSE: To evaluate the potential of proteasome inhibitors, a novel class of antitumor agents, for the treatment of retinoblastoma. The proteasome inhibitor bortezomib (PS-341, Velcade; Millennium Pharmaceuticals, Cambridge, MA), approved by the US Food and Drug Administration for the treatment of multiple myeloma, is being studied for the treatment of several other malignancies. Among other effects, it inactivates the transcription factor nuclear factor-kappaB (NF-kappaB) by blocking the degradation of its inhibitor, IkappaB. NF-kappaB, which is constitutively active in human retinoblastoma cells and promotes their survival, represents a therapeutic target for patients with this malignancy. METHODS: The authors evaluated the effect of bortezomib on the retinoblastoma cell lines Y79 and WERI-Rb1 in vitro using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, flow cytometry with propidium iodide, gene expression profiling, RT-PCR, and immunoblotting. RESULTS: Bortezomib induced caspase-dependent apoptosis in both retinoblastoma cell lines at clinically achievable concentrations. Bortezomib upregulated heat-shock proteins, other stress-response proteins, proapoptotic molecules, cell-cycle regulators, transcription factors, cytokines, and several proteasome subunits and solute carrier proteins, whereas it downregulated antiapoptotic and adhesion molecules. Bortezomib also induced cleavage of caspases, Bid and poly(ADP-ribose) polymerase (PARP), and sensitized retinoblastoma cells to doxorubicin. CONCLUSIONS: Bortezomib induces a stress response and triggers caspase-dependent apoptosis in human retinoblastoma cells at clinically achievable concentrations. This study provides insight into the molecular mechanism(s) of the antitumor activity of bortezomib and a basis for future preclinical studies leading to clinical trials of bortezomib, alone or in combination with conventional chemotherapy, to improve patient outcomes in retinoblastoma.

The role of the bone marrow microenvironment in the pathophysiology of myeloma and its significance in the development of more effective therapies.

Multiple myeloma (MM) is viewed as a prototypic disease state for the study of how neoplastic cells interact with their local bone marrow (BM) microenvironment. This interaction reflects not only the osteo-tropic clinical behavior of MM and the clinical impact of the lytic bone lesions caused by its tumor cells but also underlines the broadly accepted notion that nonneoplastic cells of the BM can attenuate the activity of cytotoxic chemotherapy and glucocorticoids. This article summarizes the recent progress in characterization, at the molecular and cellular levels, of how the BM milieu interacts with MM cells and modifies their biologic behavior.

Fas signaling in thyroid carcinomas is diverted from apoptosis to proliferation.

PURPOSE: The death receptor Fas is present in thyroid carcinomas, yet fails to trigger apoptosis. Interestingly, Fas has been reported to be actually overexpressed in papillary thyroid carcinomas, suggesting that it may confer a survival advantage. EXPERIMENTAL DESIGN: We investigated the expression and activation status of Fas pathway mediators in thyroid carcinoma cell lines and tumor specimens. RESULTS: All cell lines tested express Fas-associated death domain, procaspase-8, procaspase-9, and procaspase-3; resistance to Fas-mediated apoptosis could not be attributed to lack of any of these apoptosis mediators. Moreover, Fas death domain mutations were not found in our study. The proteasome inhibitors MG132 and PS-341 (bortezomib, Velcade), which lead to accumulation of the nuclear factor kappaB (NF-kappaB) inhibitor IkappaB, did not sensitize SW579 cells to Fas-mediated apoptosis, suggesting that resistance to Fas-mediated apoptosis is not due to proteasome or NF-kappaB activity. Cross-linking of Fas in vitro induced recruitment of Fas-associated death domain-like interleukin-1beta-converting enzyme inhibitory protein (FLIP) instead of procaspase-8. Inhibition of FLIP expression with a FLIP antisense oligonucleotide resulted in significant sensitization to Fas-mediated apoptosis. Fas cross-linking promoted BrdUrd incorporation; activated the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase/extracellular signal-regulated kinase, NF-kappaB, and activator protein-1 pathways in thyroid carcinoma cells in vitro; and protected cells from tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis. We also found that good prognosis papillary thyroid carcinoma specimens exhibited higher immunoreactivity for cleaved (activated) caspase-8 than poor prognosis tumors. CONCLUSIONS: In thyroid carcinomas, the proteolytic cleavage and activation of caspase-8 depends on the balance between expression levels for procaspase-8 and FLIP and correlates with favorable clinical prognosis. Fas may actually stimulate proliferation and confer a survival advantage to thyroid cancer cells.

Complete regression of large solid tumors using engineered drug-resistant hematopoietic cells and anti-CD137 immunotherapy.

Combining chemotherapy and immunotherapy is problematic because chemotherapy can ablate the immune responses initiated by modulators of the immune system. We hypothesized that protection of immunocompetent cells from the toxic effects of chemotherapy, using drug resistance gene therapy strategies, would allow the combined use of chemotherapy and immunotherapy. In wild-type mice, the antitumor effectiveness of an immunotherapy regimen employing an agonistic anti-CD137 antibody is diminished with escalating doses of the antifolate trimetrexate (TMTX). Using retroviral gene transfer of a mutant form of dihydrofolate reductase (L22Y-DHFR), hematopoietic stem cells were genetically engineered to withstand the toxic effects of TMTX. Mice transplanted with L22Y-DHFR-modified bone marrow were then challenged with AG104 sarcoma cells and treated with TMTX only, anti-CD137 only, or a combination of chemotherapy and immunotherapy. Although tumor burden was transiently decreased during TMTX administration, no mice treated with TMTX alone survived the tumor challenge, whereas approximately 40% of transplanted mice treated with anti-CD137 alone survived. However, 100% of mice survived with complete tumor regression after transplantation with L22Y-DHFR-transduced bone marrow followed by combined treatment with TMTX and anti-CD137. In addition, adoptive transfer of splenocytes from cured mice extended the survival of tumor- bearing animals by approximately 3 weeks compared with controls. Therefore, protection of the hematopoietic system can allow for the combined administration of chemotherapy and immunotherapy, which results in complete tumor clearance.

Antitumor effects of the proteasome inhibitor bortezomib in medullary and anaplastic thyroid carcinoma cells in vitro.

CONTEXT: The ubiquitin-proteasome pathway is a major pathway for degradation of intracellular proteins. Proteasome inhibitors constitute a novel class of antitumor agents with preclinical and clinical evidence of activity against hematological malignancies and solid tumors. The proteasome inhibitor bortezomib (PS-341, Velcade) has been approved by the Food and Drug Administration for the treatment of multiple myeloma and is being studied intensely in several other malignancies. Its mechanism of action is complex but appears to include the inhibition of inhibitory-kappaB degradation, which leads to inactivation of the transcriptional factor nuclear factor-kappaB (NF-kappaB). NF-kappaB has been implicated in the pathophysiology of the most aggressive forms of thyroid carcinoma, i.e. medullary and anaplastic. OBJECTIVE AND METHODS: We evaluated the effect of bortezomib on a panel of thyroid carcinoma cell lines, originating from papillary, follicular, anaplastic, and medullary carcinomas. RESULTS: Bortezomib induced apoptosis in medullary and anaplastic cell lines with IC(50) values well within the range of clinically achievable concentrations and much lower than respective IC(50) values for other solid malignancies. Bortezomib inhibited NF-kappaB activity; increased p53, p21, and jun expression; and induced caspase-dependent apoptosis. Sensitivity of thyroid carcinoma cells to bortezomib was partially decreased by overexpression of Bcl-2 or treatment with IGF-I, whereas the combination of bortezomib with chemotherapy (doxorubicin) was synergistic. CONCLUSIONS: These data provide both insights into the molecular mechanisms of antitumor activity of proteasome inhibitors and the rationale for future clinical trials of bortezomib, alone or in combination with conventional chemotherapy, to improve patient outcome in medullary and anaplastic thyroid carcinomas.