Eltrombopag inhibits TET dioxygenase to contribute to hematopoietic stem cell expansion in aplastic anemia.

Eltrombopag, an FDA-approved non-peptidyl thrombopoietin receptor agonist, is clinically used for the treatment of aplastic anemia, a disease characterized by hematopoietic stem cell failure and pancytopenia, to improve platelet counts and stem cell function. Eltrombopag treatment results in a durable trilineage hematopoietic expansion in patients. Some of the eltrombopag hematopoietic activity has been attributed to its off-target effects, including iron chelation properties. However, the mechanism of action for its full spectrum of clinical effects is still poorly understood. Here, we report that eltrombopag bound to the TET2 catalytic domain and inhibited its dioxygenase activity, which was independent of its role as an iron chelator. The DNA demethylating enzyme TET2, essential for hematopoietic stem cell differentiation and lineage commitment, is frequently mutated in myeloid malignancies. Eltrombopag treatment expanded TET2-proficient normal hematopoietic stem and progenitor cells, in part because of its ability to mimic loss of TET2 with simultaneous thrombopoietin receptor activation. On the contrary, TET inhibition in TET2 mutant malignant myeloid cells prevented neoplastic clonal evolution in vitro and in vivo. This mechanism of action may offer a restorative therapeutic index and provide a scientific rationale to treat selected patients with TET2 mutant-associated or TET deficiency-associated myeloid malignancies.

Therapeutic Targeting of Protein Disulfide Isomerase PDIA1 in Multiple Myeloma.

Multiple myeloma is a genetically complex hematologic neoplasia in which malignant plasma cells constantly operate at the maximum limit of their unfolded protein response (UPR) due to a high secretory burden of immunoglobulins and cytokines. The endoplasmic reticulum (ER) resident protein disulfide isomerase, PDIA1 is indispensable for maintaining structural integrity of cysteine-rich antibodies and cytokines that require accurate intramolecular disulfide bond arrangement. PDIA1 expression analysis from RNA-seq of multiple myeloma patients demonstrated an inverse relationship with survival in relapsed or refractory disease, supporting its critical role in myeloma persistence. Using a structure-guided medicinal chemistry approach, we developed a potent, orally bioavailable small molecule PDIA1 inhibitor CCF642-34. The inhibition of PDIA1 overwhelms the UPR in myeloma cells, resulting in their apoptotic cell death at doses that do not affect the normal CD34+ hematopoietic stem and progenitor cells. Bortezomib resistance leads to increased PDIA1 expression and thus CCF642-34 sensitivity, suggesting that proteasome inhibitor resistance leads to PDIA1 dependence for proteostasis and survival. CCF642-34 induces acute unresolvable UPR in myeloma cells, and oral treatment increased survival of mice in the syngeneic 5TGM1 model of myeloma. Results support development of CCF642-34 to selectively target the plasma cell program and overcome the treatment-refractory state in myeloma.

SAR insights into TET2 catalytic domain inhibition: Synthesis of 2-Hydroxy-4-Methylene-pentanedicarboxylates.

The TET (Ten-Eleven Translocation) dioxygenase enzyme family comprising 3 members, TET1-3, play key roles in DNA demethylation. These processes regulate transcription programs that determine cell lineage, survival, proliferation, and differentiation. The impetus for our investigations described here is derived from the need to develop illuminating small molecule probes for TET enzymes with cellular activity and specificity. The studies were done so in the context of the importance of TET2 in the hematopoietic system and the preponderance of loss of function somatic TET2 mutations in myeloid diseases. We have identified that 2-hydroxy-4-methylene-pentanedicarboxylic acid 2a reversibly competes with the co-substrate α-KG in the TET2 catalytic domain and inhibits the dioxygenase activity with an IC50 = 11.0 ± 0.9 µM at 10 µM α-KG in a cell free system and binds in the TET2 catalytic domain with Kd = 0.3 ± 0.12 µM.

A Therapeutic Strategy for Preferential Targeting of TET2 Mutant and TET-dioxygenase Deficient Cells in Myeloid Neoplasms.

TET2 is frequently mutated in myeloid neoplasms. Genetic TET2 deficiency leads to skewed myeloid differentiation and clonal expansion, but minimal residual TET activity is critical for survival of neoplastic progenitor and stem cells. Consistent with mutual exclusivity of TET2 and neomorphic IDH1/2 mutations, here we report that IDH1/2 mutant-derived 2-hydroxyglutarate is synthetically lethal to TET-dioxygenase deficient cells. In addition, a TET-selective small molecule inhibitor decreased cytosine hydroxymethylation and restricted clonal outgrowth of TET2 mutant, but not normal hematopoietic precursor cells in vitro and in vivo. While TET-inhibitor phenocopied somatic TET2 mutations, its pharmacologic effects on normal stem cells were, unlike mutations, reversible. Treatment with TET inhibitor suppressed the clonal evolution of TET2 mutant cells in murine models and TET2-mutated human leukemia xenografts. These results suggest that TET inhibitors may constitute a new class of targeted agents in TET2 mutant neoplasia.

DNA methylation inhibition in myeloma: Experience from a phase 1b study of low-dose continuous azacitidine in combination with lenalidomide and low-dose dexamethasone in relapsed or refractory multiple myeloma.

The DNA methyltransferase inhibitor azacytidine (aza) may reactivate pathways associated with plasma cell differentiation, cell cycle control, apoptosis, and immune recognition and thereby restore sensitivity to lenalidomide (len) and dexamethasone (dex) in relapsed and/or refractory multiple myeloma (RRMM). We aimed to develop an aza regimen that reaches epigenetically active levels 8 times in 28 days with less bone marrow toxicity than the myeloid malignancy standard of 7 consecutive doses to enable safe combination with len. Aza was escalated from 30 mg/m2 once a week up to a predefined maximum of 50 mg/m2 twice a week in combination with GFR-adjusted len (≥ 60 mL/min: 25 mg, 3059 mL/min: 10 mg) day 1 to 21 every 28 days and dex 40 mg once a week followed by a limited expansion study to a total N of 23 at the highest tolerated dose. Fifty-one patients (pts) with RRMM were screened, 42 were treated and 41 were evaluable for response based on at least 1 response assessment or progression after treatment start. The median number of prior lines of therapy was 5 (1-11) and 81% (34) were refractory to len and/or pomalidomide (pom). Two DLTs occurred in different cohorts, 1 neutropenic fever in 1/6 pts on the aza 40 mg/m2 twice a week GFR ≥ 60 mL/min cohort and 1 GGT elevation in 1/6 pts on the aza 50 mg/m2 GFR 30-59 mL/min cohort. An MTD was not reached and aza 50 mg/m2 SC twice a week was chosen for the expansion study. At least possibly related Grade 3/4 AEs occurred in 28 pts (67%) with the following in > 1 pt: neutropenia (N = 16, 38%), anemia (N = 6, 14%), lymphopenia (N = 5, 12%), thrombocytopenia (N = 4, 10%), leukopenia (N = 4, 10%), febrile neutropenia (N = 4, 10%), fatigue (N = 3, 7%), fever (N = 2, 5%), and infection (N = 2, 5%). At a median follow up time for alive pts of 60.2 months (range: 36.1-82.5 months), the overall response rate (≥ partial response) and clinical benefit response rate (≥ minor response) was 22 and 32%, respectively, with 4 very good partial responses (10%), 5 partial responses (12%), and 4 minor responses (10%). The median PFS was 3.1 months (95% confidence interval [CI]: 2.1-5.1 months), median TTP 2.7 months (95% CI: 2.1-7.5 months), and median OS 18.6 months (95% CI: 12.9-33.0 months). Achieving at least minor response and reaching TTP > 6 months was associated with approximately 35% lower median plasma levels of the enzyme that inactivates aza, plasma cytidine deaminase (CDA, P< .0001). Two of the len refractory pts achieved longer disease control than with any prior regimen and 1 responded immediately after progression on len, bortezomib, and prednisone. Analyses of the methylation state of over 480,000 CpG sites in purified myeloma cells at screening were possible in 11 pts and on day 28 in 8 of them. As in other studies, the majority of differentially methylated CpGs compared to normal plasma cells were hypomethylated in myeloma. Treatment decreased the number of CpGs that were differentially methylated in normal plasma cells by > 0.5% in 6 and by > 5% in 3 of the 8 pts, most pronounced in 2 pts with clinically convincing aza contribution who achieved a reduction in overall differentially methylated CpGs by 23 and 68%, respectively, associated with increased expression of immunoglobulin genes. The study demonstrated tolerability of twice a week SC aza at 50 mg/m2 with len and dex in RRMM and suggested aza may help overcome the len/pom refractory state, possibly by activating differentiation pathways. Relatively low response rates and association of clinical benefit with low plasma levels of the aza inactivating enzyme CDA suggest the aza regimen will need to be optimized further and pt selection may be required to maximize benefit.

Decitabine- and 5-azacytidine resistance emerges from adaptive responses of the pyrimidine metabolism network.

Mechanisms-of-resistance to decitabine and 5-azacytidine, mainstay treatments for myeloid malignancies, require investigation and countermeasures. Both are nucleoside analog pro-drugs processed by pyrimidine metabolism into a deoxynucleotide analog that depletes the key epigenetic regulator DNA methyltranseferase 1 (DNMT1). Here, upon serial analyses of DNMT1 levels in patients' bone marrows on-therapy, we found DNMT1 was not depleted at relapse. Showing why, bone marrows at relapse exhibited shifts in expression of key pyrimidine metabolism enzymes in directions adverse to pro-drug activation. Further investigation revealed the origin of these shifts. Pyrimidine metabolism is a network that senses and regulates deoxynucleotide amounts. Deoxynucleotide amounts were disturbed by single exposures to decitabine or 5-azacytidine, via off-target depletion of thymidylate synthase and ribonucleotide reductase respectively. Compensating pyrimidine metabolism shifts peaked 72-96 h later. Continuous pro-drug exposures stabilized these adaptive metabolic responses to thereby prevent DNMT1-depletion and permit exponential leukemia out-growth as soon as day 40. The consistency of the acute metabolic responses enabled exploitation: simple treatment modifications in xenotransplant models of chemorefractory leukemia extended noncytotoxic DNMT1-depletion and leukemia control by several months. In sum, resistance to decitabine and 5-azacytidine originates from adaptive responses of the pyrimidine metabolism network; these responses can be anticipated and thus exploited.

Roles of the C-terminal domains of topoisomerase IIα and topoisomerase IIß in regulation of the decatenation checkpoint.

Topoisomerase (topo) IIα and IIß maintain genome stability and are targets for anti-tumor drugs. In this study, we demonstrate that the decatenation checkpoint is regulated, not only by topo IIα, as previously reported, but also by topo IIß. The decatenation checkpoint is most efficient when both isoforms are present. Regulation of this checkpoint and sensitivity to topo II-targeted drugs is influenced by the C-terminal domain (CTD) of the topo II isoforms and by a conserved non-catalytic tyrosine, Y640 in topo IIα and Y656 in topo IIß. Deletion of most of the CTD of topo IIα, while preserving the nuclear localization signal (NLS), enhances the decatenation checkpoint and sensitivity to topo II-targeted drugs. In contrast, deletion of most of the CTD of topo IIß, while preserving the NLS, and mutation of Y640 in topo IIα and Y656 in topo IIß inhibits these activities. Structural studies suggest that the differential impact of the CTD on topo IIα and topo IIß function may be due to differences in CTD charge distribution and differential alignment of the CTD with reference to transport DNA. Together these results suggest that topo IIα and topo IIß cooperate to maintain genome stability, which may be distinctly modulated by their CTDs.

Novel Protein Disulfide Isomerase Inhibitor with Anticancer Activity in Multiple Myeloma.

Multiple myeloma cells secrete more disulfide bond-rich proteins than any other mammalian cell. Thus, inhibition of protein disulfide isomerases (PDI) required for protein folding in the endoplasmic reticulum (ER) should increase ER stress beyond repair in this incurable cancer. Here, we report the mechanistically unbiased discovery of a novel PDI-inhibiting compound with antimyeloma activity. We screened a 30,355 small-molecule library using a multilayered multiple myeloma cell-based cytotoxicity assay that modeled disease niche, normal liver, kidney, and bone marrow. CCF642, a bone marrow-sparing compound, exhibited a submicromolar IC50 in 10 of 10 multiple myeloma cell lines. An active biotinylated analog of CCF642 defined binding to the PDI isoenzymes A1, A3, and A4 in MM cells. In vitro, CCF642 inhibited PDI reductase activity about 100-fold more potently than the structurally distinct established inhibitors PACMA 31 and LOC14. Computational modeling suggested a novel covalent binding mode in active-site CGHCK motifs. Remarkably, without any further chemistry optimization, CCF642 displayed potent efficacy in an aggressive syngeneic mouse model of multiple myeloma and prolonged the lifespan of C57BL/KaLwRij mice engrafted with 5TGM1-luc myeloma, an effect comparable to the first-line multiple myeloma therapeutic bortezomib. Consistent with PDI inhibition, CCF642 caused acute ER stress in multiple myeloma cells accompanied by apoptosis-inducing calcium release. Overall, our results provide an illustration of the utility of simple in vivo simulations as part of a drug discovery effort, along with a sound preclinical rationale to develop a new small-molecule therapeutic to treat multiple myeloma. Cancer Res; 76(11); 3340-50. ©2016 AACR.

Inhibition of proteasome activity by bortezomib in renal cancer cells is p53 dependent and VHL independent.

BACKGROUND: Antiproliferative effects of proteasome inhibitors are suggested to be primarily due to effects on nuclear factor-kappaB (NF-kappaB)-dependent pathways and the induction of apoptosis. The objective of this study was to elucidate the mechanistic basis for the antiproliferative effects of the proteasome inhibitor, bortezomib, in human clear cell renal cell cancer cells (CCRCC). MATERIALS AND METHODS: von Hippel Lindau (VHL) mutation/methylation status and cytotoxic response to bortezomib was determined in a panel of CCRCC cell lines. Effects on target protein/gene expression and the role of p53 in bortezomib-mediated cytotoxicity, inhibition of proteasome activity, survivin transcript and protein expression as well as induction of p21 expression was determined in CCRCC that differed in their intrinsic sensitivity to bortezomib. RESULTS: VHL status was not associated with cytotoxic response to bortezomib treatment. Cytotoxicity in cell lines that differed in intrinsic sensitivity to bortezomib correlated with sustained inhibition of proteasome activity, survivin expression and induction of p21 expression. Stable down-regulation of p53 expression by siRNA led to attenuation of bortezomib effects, survivin down-regulation and p21 induction, suggesting that cellular effects are p53-dependent. CONCLUSION: These results demonstrate that the antiproliferative effects of bortezomib in CCRCC cells are VHL independent and dependent on pathways regulated by p53.

Casein kinase I delta/epsilon phosphorylates topoisomerase IIalpha at serine-1106 and modulates DNA cleavage activity.

We previously reported that phosphorylation of topoisomerase (topo) IIalpha at serine-1106 (Ser-1106) regulates enzyme activity and sensitivity to topo II-targeted drugs. In this study we demonstrate that phosphorylation of Ser-1106, which is flanked by acidic amino acids, is regulated in vivo by casein kinase (CK) Idelta and/or CKIepsilon, but not by CKII. The CKI inhibitors, CKI-7 and IC261, reduced Ser-1106 phosphorylation and decreased formation of etoposide-stabilized topo II-DNA cleavable complex. In contrast, the CKII inhibitor, 5,6-dichlorobenzimidazole riboside, did not affect etoposide-stabilized topo II-DNA cleavable complex formation. Since, IC261 specifically targets the Ca(2+)-regulated isozymes, CKIdelta and CKIepsilon, we examined the effect of down-regulating these enzymes on Ser-1106 phosphorylation. Down-regulation of these isozymes with targeted si-RNAs led to hypophosphorylation of the Ser-1106 containing peptide. However, si-RNA-mediated down-regulation of CKIIalpha and alpha' did not alter Ser-1106 phosphorylation. Furthermore, reduced phosphorylation of Ser-1106, observed in HRR25 (CKIdelta/epsilon homologous gene)-deleted Saccharomyces cerevisiae cells transformed with human topo IIalpha, was enhanced following expression of human CKIepsilon. Down-regulation of CKIdelta and CKIepsilon also led to reduced formation of etoposide stabilized topo II-DNA cleavable complex. These results provide strong support for an essential role of CKIdelta/epsilon in phosphorylating Ser-1106 in human topo IIalpha and in regulating enzyme function.

Proteasome inhibition with bortezomib enhances activity of topoisomerase I-targeting drugs by NF-kappaB-independent mechanisms.

PURPOSE: The potentiation of topoisomerase (topo)-I-induced apoptosis by proteasome inhibitors is dependent on the treatment sequence, but not on NF-kappaB. In this study, alternate mechanisms modulating apoptosis induced with the topo I-targeting drug, SN-38, when followed by the proteasome inhibitor bortezomib (PS-341) were investigated. MATERIALS AND METHODS: Human non-small cell lung carcinoma (NSCLC-3) cells transfected with a control vector (NSCLC-3/neo) or a vector containing dominant negative IkappaBalpha (NSCLC-3/mIkappaBalpha) were treated with SN-38 for 1 h followed by PS-341 for 4 h (SN-38 --> PS-341), or with either drug alone. The functional role of the anti-apoptotic protein survivin was tested using NSCLC-3 transfected with myc-tagged wild-type (NSCLC-3/myc-survivin), or dominant negative mutant T34A survivin (NSCLC-3/myc-T34A). RESULTS: In NSCLC-3/neo or NSCLC-3/mIkappaBalpha cells, treatment with SN-38 --> PS-341 led to down-regulation of the survivin transcript and protein, enhanced apoptosis and reduced (> 3-fold) survival compared to SN-38 or PS-341 alone. In contrast to the cells transfected with wild-type survivin, or the control NSCLC-3/neo, those cells transfected with mutant survivin and treated with SN-38 --> PS-341 exhibited enhanced caspase 9 activity (> 2-fold), caspase 3 (> 2- to 3-fold) activity and cytotoxicity compared to the NSCLC-3/neo cells. CONCLUSION: In contrast to inhibition of NF-kappaB activity, down-regulation of the anti-apoptotic survivin was correlated with modulation of the sequence-dependent synergistic effects of PS-341 in SN-38-induced apoptosis.

Sensitization of DNA damage-induced apoptosis by the proteasome inhibitor PS-341 is p53 dependent and involves target proteins 14-3-3sigma and survivin.

Proteasome inhibition following DNA damage results in the synergistic induction of apoptosis via a nuclear factor-kappaB-independent mechanism. In this study, we identify the role of p53 in mediating apoptosis by the sequence-specific treatment involving the DNA-damaging, topoisomerase I-targeting drug SN-38 followed by the proteasome inhibitor PS-341 (SN-38-->PS-341). The p53-dependent sensitization of DNA damage-induced apoptosis by PS-341 is accompanied by persistent inhibition of proteasome activity and increased cytosolic accumulation of p53, including higher molecular weight forms likely representing ubiquitinated species. In contrast, pretreatment with PS-341 followed by treatment with SN-38 (PS-341-->SN-38), which leads to an antagonistic interaction, results in transient inhibition of proteasome activity and accumulation of significantly lower levels of p53 localized primarily to the nucleus. Whereas cells treated with PS-341-->SN-38 undergo G2 + M cell cycle arrest, cells treated with SN-38-->PS-341 exhibit a decreased G2 + M block with a concomitant increase in the sub-G1 population. Decreased accumulation of cells in the G2 + M phase of the cell cycle in SN-38-->PS-341-treated cells compared with PS-341-->SN-38-treated cells correlates with enhanced apoptosis and reduced expression of two p53-modulated proteins, 14-3-3sigma and survivin, both of which play critical roles in regulating G2 + M progression and apoptosis. The functional role of 14-3-3sigma or survivin in regulating the divergent function of p53 in response to SN-38-->PS-341 and PS-341-->SN-38 treatment in inducing apoptosis versus G2 + M arrest/DNA repair, respectively, was confirmed by targeted down-regulation of these proteins. These results provide insights into the mechanisms by which inhibition of proteasome activity modulates DNA damage-induced apoptosis via a p53-dependent pathway.

c-IAP1 is overexpressed in HL-60 cells selected for doxorubicin resistance: effects on etoposide-induced apoptosis.

BACKGROUND: We previously demonstrated that KN-62, an inhibitor of calcium calmodulin-dependent enzymes, sensitizes human leukemia HL-60 cells resistant to topoisomerase II-targeting drugs. The objective of this study was to determine pathways of apoptosis downstream of DNA damage induced by KN-62 co-treatment with VP-16. MATERIALS AND METHODS: HL-60/Y/DOX0.05 cells were treated with VP-16, KN-62, or VP-16 + KN-62. Following treatment, cells were assayed for c-IAP1, c-IAP2 and XIAP protein expression, as well as caspase activation, cytochrome c release and PARP cleavage. RESULTS: Baseline c-IAP1 protein levels were 2-fold higher in HL60 cells selected for resistance to doxorubicin compared to the parent sensitive line. VP-16 and KN-62 co-treatment was associated with caspase activation via the mitochondrial pathway and significant reductions (p = 0.002) in c-IAP1 protein expression but not with c-IAP2 or XIAP. CONCLUSION: These data suggest that KN-62 co-treatment sensitizes doxorubicin-resistant cells to VP-16-induced apoptosis by enhancing caspase activity and reducing c-IAP1 expression.

Apoptotic pathways of epothilone BMS 310705.

OBJECTIVE: BMS 310705 is a novel water-soluble analog of epothilone B currently in phase I clinical evaluation in the treatment of malignancies such as ovarian, renal, bladder, and lung carcinoma. Using an early passage cell culture model derived from the ascites of a patient clinically refractory to platinum/paclitaxel therapy, we evaluated the pathway of caspase-mediated apoptosis. METHODS: Cells were treated for 1 h and subsequently evaluated for apoptosis, survival, and caspase activity. Apoptosis was determined by fluorescent microscopy. Caspase-3, -8, and -9 activities were determined by fluorometry using target tetrapeptide substrates. Mitochondrial release of cytochrome c was determined by immunoblot analysis. RESULTS: After treatment with BMS 310705, apoptosis was confirmed in >25% of cells at 24 h. Survival was significantly lower (P < 0.02) in cells treated with 0.05 micro M BMS 310705 vs paclitaxel. Analysis revealed an increase of caspase-9 and -3 activity; no caspase -8 activity was observed. Release of cytochrome c was detected at 12 h following treatment. SN-38 and topotecan failed to induce apoptosis. CONCLUSIONS: BMS 310705 induces significant apoptosis, decreases survival, and utilizes the mitochondrial-mediated pathway for apoptosis in this model.

Phosphorylation of serine 1106 in the catalytic domain of topoisomerase II alpha regulates enzymatic activity and drug sensitivity.

Topoisomerases alter DNA topology and are vital for the maintenance of genomic integrity. Topoisomerases I and II are also targets for widely used antitumor agents. We demonstrated previously that in the human leukemia cell line, HL-60, resistance to topoisomerase (topo) II-targeting drugs such as etoposide is associated with site-specific hypophosphorylation of topo II alpha. This effect can be mimicked in sensitive cells treated with the intracellular Ca(2+) chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM). Here we identify Ser-1106 as a major phosphorylation site in the catalytic domain of topo II alpha. This site lies within the consensus sequence for the acidotrophic kinases, casein kinase I and casein kinase II. Mutation of serine 1106 to alanine (S1106A) abrogates phosphorylation of phosphopeptides that were found to be hypophosphorylated in resistant HL-60 cells or sensitive cells treated with BAPTA-AM. Purified topo II alpha containing a S1106A substitution is 4-fold less active than wild type topo II alpha in decatenating kinetoplast DNA and also exhibits a 2-4-fold decrease in the level of etoposide-stabilized DNA cleavable complex formation. Saccharomyces cerevisiae (JN394t2-4) cells expressing S1106A mutant topo II alpha protein are more resistant to the cytotoxic effects of etoposide or amsacrine. These results demonstrate that Ca(2+)-regulated phosphorylation of Ser-1106 in the catalytic domain of topo II alpha modulates the enzymatic activity of this protein and sensitivity to topo II-targeting drugs.

Inhibition of NF-kappaB and proteasome activity in tumors: can we improve the therapeutic potential of topoisomerase I and topoisomerase II poisons.

Activation of signaling pathways following DNA damage induced by topoisomerase (topo) poisons can lead to cell death by apoptosis. NF-kappaB, a major regulator of the stress response and a negative regulator of apoptosis is often activated following treatment with topoisomerase poisons. Since activation of NF-kappaB is generally considered to relay an anti-apoptotic signal, inactivation of this signaling molecule is considered to represent an important strategy to improve therapeutic efficacy. Although this strategy seems to be effective in some model systems, our results in human non-small cell lung cancers differed. In this review we will discuss the role of NF-kappaB in mediating topoisomerase poison-induced DNA damage and apoptosis and the consequence of inhibiting its activity. Newer insights about the importance of proteasome inhibitors and anti-apoptotic genes in topoisomerase poison-induced signaling mechanisms leading to apoptosis will also be reviewed. The knowledge obtained from these studies may be useful for translation to a clinical setting for development of more effective therapeutic strategies.