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We report the results of a phase 1 dose-escalation study of belinostat and bortezomib in adult patients with acute leukemia or MDS or CML with blast crisis. Thirty-eight patients received IV belinostat days 1-5 and 8-12 with IV bortezomib days 1, 4, 8, and 11 every 21 days. QTc prolongation was the only identified DLT. The RP2Ds were 1.3 mg/m 2 bortezomib and 1000 mg/m 2 belinostat. One patient with highly refractory MLL-ENL rearranged biphenotypic AML with multiple karyotypic aberrations had a complete pathologic and karyotypic response. One patient with post-MPN AML remained on study with stable disease (SD) for 32 cycles. Whole-exome sequencing revealed no aberrations in the first patient and a hyper-mutator genotype in the second. Eighteen patients had a best response of SD. We conclude that this treatment strategy is feasible but has limited activity in this population. Nevertheless, the factors that predict exceptional responses to this strategy warrant further investigation.

Introduction: Targeting anti-apoptotic pathways involving the BCL2 family proteins represents a novel treatment strategy in hematologic malignancies. Venetoclax, a selective BCL2 inhibitor, represents the first approved agent of this class, and is currently used in CLL and AML. However, monotherapy is rarely sufficient for sustained responses due to the development of drug resistance and loss of dependence upon the targeted protein. Numerous pre-clinical studies have shown that combining venetoclax with other agents may represent a more effective therapeutic strategy by circumventing resistance mechanisms. In this review, we summarize pre-clinical data providing a foundation for rational combination strategies involving venetoclax., Areas Covered: Novel combination strategies in hematologic malignancies involving venetoclax, primarily at the pre-clinical level, will be reviewed. We emphasize novel agents that interrupt complementary or compensatory pro-survival pathways, and particularly mechanistic insights underlying synergism. PubMed, Cochrane, EMBASE, and Google scholar were searched from 2000., Expert Opinion: Although venetoclax has proven to be an effective therapeutic in hematologic malignancies, monotherapy may be insufficient for maximal effectiveness due to the development of resistance and/or loss of BCL2 addiction. Further pre-clinical and clinical development of combination therapies may be necessary for optimal outcomes in patients with diverse blood cancers.

Venetoclax (ABT-199) is a Bcl-2-specific BH3-mimetic that has shown significant promise in certain subtypes of CLL as well as in several other hematologic malignancies. As in the case of essentially all targeted agents, intrinsic or acquired resistance to this agent generally occurs, prompting the search for new strategies capable of circumventing this problem. A logical approach to this challenge involves rational combination strategies designed to disable preexisting or induced compensatory survival pathways. Many of these strategies involve downregulation of Mcl-1, a pro-survival Bcl-2 family member that is not targeted by venetoclax, and which often confers resistance to this agent. Given encouraging clinical results involving venetoclax in both lymphoid and myeloid malignancies, it is likely that such combination approaches will be incorporated into the therapeutic armamentarium for multiple hematologic malignancies in the near future.

A phase 1 study was conducted to determine the dose-limiting toxicities and maximum-tolerated dose (MTD) for bortezomib followed by romidepsin on days 1, 8, and 15 in patients with relapsed/refractory CLL/SLL or B- or T-cell lymphoma. Eighteen treated patients were evaluable for response. The MTD was 1.3 mg/m 2 bortezomib and 10 mg/m 2 romidepsin; median treatment duration was 3 cycles at this dose. The dose-limiting toxicities were grade 3 fatigue, vomiting, and chills. Two patients had partial responses, one lasting >2 years, 8 had stable disease, and 8 had progressive disease. The median duration of stable disease was 3.5 cycles. Correlative studies examining expression of NF-кB, XIAP, Bcl-xL, and Bim yielded variable results. The safety profile was consistent with that reported for single-agent bortezomib and romidepsin. This regimen has modest activity in heavily pretreated patients with relapsed/refractory CLL or B- or T-cell lymphoma. NCT00963274.

A phase 1 study with carfilzomib and vorinostat was conducted in 20 B-cell lymphoma patients. Vorinostat was given orally twice daily on days 1, 2, 3, 8, 9, 10, 15, 16, and 17 followed by carfilzomib (given as a 30-min infusion) on days 1, 2, 8, 9, 15, and 16. A treatment cycle was 28 days. Dose escalation initially followed a standard 3 + 3 design, but adapted a more conservative accrual rule following dose de-escalation. The maximum tolerated dose was 20 mg/m2 carfilzomib and 100 mg vorinostat (twice daily). The dose-limiting toxicities were grade 3 pneumonitis, hyponatremia, and febrile neutropenia. One patient had a partial response and two patients had stable disease. Correlative studies showed a decrease in NF-κB activation and an increase in Bim levels in some patients, but these changes did not correlate with clinical response.

Introduction: Patients with relapsed or refractory lymphoma remain a population with unmet medical needs. Histone deacetylase inhibitors (HDACIs) represent a novel class of anticancer drugs currently in development in several malignancies. Inhibition of HDACs leads to acetylation of histone and non-histone proteins, which in turn results in epigenetic modification of gene expression that leads to a plethora of effects, such as cell cycle arrest, apoptosis and inhibition of angiogenesis. Romidepsin is a novel HDACI that has demonstrated preclinical and clinical activity., Areas Covered: This review discusses the different HDACs and epigenetic regulation with a particular focus on the preclinical and clinical development of romidepsin in lymphoma. The review of romidepsin includes: the mechanism of action, its synergistic interaction with novel agents, pivotal clinical trials that lead to its US FDA approval in cutaneous T-cell lymphoma and peripheral T-cell lymphoma as well as active combinations currently in clinical trials., Expert Opinion: Romidepsin is a potent HDACI with clinical activity in T-cell lymphoma where novel agents and combinations are desperately needed. A deeper understanding of the molecular characteristics of this class of agents will allow the design of more potent drugs with improved toxicity profiles and future rational combinations that will expand the indication and benefit from these novel agents.

A variety of anticancer agents employed in standard chemotherapy or novel targeted therapy induce autophagy. A cytoprotective autophagic response often counteracts apoptosis triggered by such agents, potentially contributing to acquired drug-resistance. It is recognized that autophagy and apoptosis share molecular regulatory mechanisms primarily governed by multidomain anti-apoptotic members (e.g., BCL2/Bcl(-)2 and BCL2L1/Bcl(-)xL) of the BCL2 family. However, the role of pro-apoptotic BH3-only proteins (e.g.,, BCL2L11/Bim), another class of BCL2 family proteins that critically determine therapeutic responses, in autophagy regulation remains largely unexplored, particularly with respect to mechanisms of acquired drug resistance.

In selective autophagy, the adaptor protein SQSTM1/p62 plays a critical role in recognizing/loading cargo (e.g., malfolded proteins) into autophagosomes for lysosomal degradation. Here we report that whereas SQSTM1/p62 levels fluctuated in a time-dependent manner during autophagy, inhibition or knockdown of Cdk9/cyclin T1 transcriptionally downregulated SQSTM1/p62 but did not affect autophagic flux. These interventions, or short hairpin RNA (shRNA) directly targeting SQSTM1/p62, resulted in cargo loading failure and inefficient autophagy, phenomena recently described for Huntington's disease neurons. These events led to the accumulation of the BH3-only protein NBK/Bik on endoplasmic reticulum (ER) membranes, most likely by blocking loading and autophagic degradation of NBK/Bik, culminating in apoptosis. Whereas NBK/Bik upregulation was further enhanced by disruption of distal autophagic events (e.g., autophagosome maturation) by chloroquine (CQ) or Lamp2 shRNA, it was substantially diminished by inhibition of autophagy initiation (e.g., genetically by shRNA targeting Ulk1, beclin-1, or Atg5 or pharmacologically by 3-methyladenine [3-MA] or spautin-1), arguing that NBK/Bik accumulation stems from inefficient autophagy. Finally, NBK/Bik knockdown markedly attenuated apoptosis in vitro and in vivo. Together, these findings identify novel cross talk between autophagy and apoptosis, wherein targeting SQSTM1/p62 converts cytoprotective autophagy to an inefficient form due to cargo loading failure, leading to NBK/Bik accumulation, which triggers apoptosis., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

We determined whether clinically relevant phosphodiesterase 5 (PDE5) inhibitors interacted with clinically relevant chemotherapies to kill medulloblastoma cells. In medulloblastoma cells PDE5 inhibitors interacted in a greater than additive fashion with vincristine/etoposide/cisplatin to cause cell death. Knockdown of PDE5 expression recapitulated the combination effects of PDE5 inhibitor drugs with chemotherapy drugs. Expression of dominant negative caspase 9 did not significantly inhibit chemotherapy lethality but did significantly reduce enhanced killing in combination with the PDE5 inhibitor sildenafil. Overexpression of BCL-XL and c-FLIP-s suppressed individual and combination drug toxicities. Knockdown of CD95 or FADD suppressed drug combination toxicity. Treatment with PDE5 inhibitors and chemotherapy drugs promoted autophagy which was maximal at ~12 h post-treatment, and in a cell type-dependent manner knockdown of Beclin1 or ATG5 either suppressed or enhanced drug combination lethality. PDE5 inhibitors enhanced the induction of chemotherapy-induced DNA damage in a nitric oxide synthase-dependent fashion. In conclusion, our data demonstrate that the combination of PDE5 inhibitors with standard of care chemotherapy agents for medulloblastoma represents a possible novel modality for future treatment of this disease.

The present studies were to determine whether the multi-kinase inhibitor pazopanib interacted with histone deacetylase inhibitors (HDACI: valproate, vorinostat) to kill sarcoma cells. In multiple sarcoma cell lines, at clinically achievable doses, pazopanib and HDACI interacted in an additive to greater than additive fashion to cause tumor cell death. The drug combination increased the numbers of LC3-GFP and LC3-RFP vesicles. Knockdown of Beclin1 or ATG5 significantly suppressed drug combination lethality. Expression of c-FLIP-s, and to a lesser extent BCL-XL or dominant negative caspase 9 reduced drug combination toxicity; knock down of FADD or CD95 was protective. Expression of both activated AKT and activated MEK1 was required to strongly suppress drug combination lethality. The drug combination inactivated mTOR and expression of activated mTOR strongly suppressed drug combination lethality. Treatment of animals carrying sarcoma tumors with pazopanib and valproate resulted in a greater than additive reduction in tumor volume compared with either drug individually. As both pazopanib and HDACIs are FDA-approved agents, our data argue for further determination as to whether this drug combination is a useful sarcoma therapy in the clinic.

The present studies determined whether the antibiotic salinomycin interacted with HDAC inhibitors to kill primary human GBM cells. Regardless of PTEN, ERBB1, or p53 mutational status salinomycin interacted with HDAC inhibitors in a synergistic fashion to kill GBM cells. Inhibition of CD95/Caspase 8 or of CD95/RIP-1/AIF signaling suppressed killing by the drug combination. Salinomycin increased the levels of autophagosomes that correlated with increased p62 and LC3II levels; valproate co-treatment correlated with reduced LC3II and p62 expression, and increased caspase 3 cleavage. Molecular inhibition of autophagosome formation was protective against drug exposure. The drug combination enhanced eIF2α phosphorylation and decreased expression of MCL-1 and phosphorylation of mTOR and p70 S6K. Activation of p70 S6K or mTOR promoted cell survival in the face of combined drug exposure. Overexpression of BCL-XL or c-FLIP-s was protective. Collectively our data demonstrate that the lethality of low nanomolar concentrations of salinomycin are enhanced by HDAC inhibitors in GBM cells and that increased death receptor signaling together with reduced mitochondrial function are causal in the combinatorial drug necro-apoptotic killing effect.

In the present study we show that histone deacetylase inhibitors (HDACIs) enhance the anti-tumor effects of melanoma differentiation associated gene-7/interleukin 24 (mda- 7/IL-24) in human renal carcinoma cells. Similar data were obtained in other GU tumor cells. Combination of these two agents resulted in increased autophagy that was dependent on expression of ceramide synthase 6, with HDACIs enhancing MDA-7/IL-24 toxicity by increasing generation of ROS and Ca (2+). Knock down of CD95 protected cells from HDACI and MDA-7/IL-24 lethality. Sorafenib treatment further enhanced (HDACI + MDA-7/IL-24) lethality. Anoikis resistant renal carcinoma cells were more sensitive to MDA-7/IL-24 that correlated with elevated SRC activity and tyrosine phosphorylation of CD95. We employed a recently constructed serotype 5/3 adenovirus, which is more effective than a serotype 5 virus in delivering mda- 7/IL-24 to renal carcinoma cells and which conditionally replicates (CR) in tumor cells expressing MDA-7/IL-24 by virtue of placing the adenoviral E1A gene under the control of the cancer-specific promoter progression elevated gene-3 (Ad.5/3-PEG-E1A-mda-7; CRAd.5/3-mda-7, Ad.5/3-CTV), to define efficacy in renal carcinoma cells. Ad.5/3-CTV decreased the growth of renal carcinoma tumors to a significantly greater extent than did a non-replicative virus Ad.5/3-mda-7. In contralateral uninfected renal carcinoma tumors Ad.5/3-CTV also decreased the growth of tumors to a greater extent than did Ad.5/3-mda-7. In summation, our data demonstrates that HDACIs enhance MDA-7/IL-24-mediated toxicity and tumor specific adenoviral delivery and viral replication of mda-7/IL-24 is an effective pre-clinical renal carcinoma therapeutic.

The present studies focused on defining the mechanisms by which anoikis-resistant (AR) mammary carcinoma cells can be reverted to a therapy-sensitive phenotype. AR mammary carcinoma cells had reduced expression of the toxic BH3 domain proteins BAX, BAK, NOXA, and PUMA. In AR cells expression of the protective BCL-2 family proteins BCL-XL and MCL-1 was increased. AR cells were resistant to cell killing by multiple anti-tumor cell therapies, including ERBB1/2 inhibitor + MCL-1 inhibitor treatment, and had a reduced autophagic flux response to these therapies, despite similarly exhibiting increased levels of LC3II processing. Knockdown of MCL-1 and BCL-XL caused necro-apoptosis in AR cells to a greater extent than in parental cells. Pre-treatment of anoikis-resistant cells with histone deacetylase inhibitors (HDACIs) for 24 h increased the levels of toxic BH3 domain proteins, reduced MCL-1 levels, and restored/re-sensitized the cell death response of AR tumor cells to multiple toxic therapies. In vivo, pre-treatment of AR breast tumors in the brain with valproate restored the chemo-sensitivity of the tumors and prolonged animal survival. These data argue that one mechanism to enhance the anti-tumor effect of chemotherapy could be HDACI pre-treatment.

Introduction: Cyclin-dependent kinases (CDKs) regulate cell cycle progression. Certain CDKs (e.g., CDK7, CDK9) also control cellular transcription. Consequently, CDKs represent attractive targets for anticancer drug development, as their aberrant expression is common in diverse malignancies, and CDK inhibition can trigger apoptosis. CDK inhibition may be particularly successful in hematologic malignancies, which are more sensitive to inhibition of cell cycling and apoptosis induction., Areas Covered: A number of CDK inhibitors, ranging from pan-CDK inhibitors such as flavopiridol (alvocidib) to highly selective inhibitors of specific CDKs (e.g., CDK4/6), such as PD0332991, that are currently in various phases of development, are profiled in this review. Flavopiridol induces cell cycle arrest, and globally represses transcription via CDK9 inhibition. The latter may represent its major mechanism of action via down-regulation of multiple short-lived proteins. In early phase trials, flavopiridol has shown encouraging efficacy across a wide spectrum of hematologic malignancies. Early results with dinaciclib and PD0332991 also appear promising., Expert Opinion: In general, the antitumor efficacy of CDK inhibitor monotherapy is modest, and rational combinations are being explored, including those involving other targeted agents. While selective CDK4/6 inhibition might be effective against certain malignancies, broad-spectrum CDK inhibition will likely be required for most cancers.

The present studies examined viability and DNA damage levels in mammary carcinoma cells following PARP1 and CHK1 inhibitor drug combination exposure. PARP1 inhibitors [AZD2281 ; ABT888 ; NU1025 ; AG014699] interacted with CHK1 inhibitors [UCN-01 ; AZD7762 ; LY2603618] to kill mammary carcinoma cells. PARP1 and CHK1 inhibitors interacted to increase both single strand and double strand DNA breaks that correlated with increased γH2AX phosphorylation. Treatment of cells with CHK1 inhibitors increased the phosphorylation of CHK1 and ERK1/2. Knock down of ATM suppressed the drug-induced increases in CHK1 and ERK1/2 phosphorylation and enhanced tumor cell killing by PARP1 and CHK1 inhibitors. Expression of dominant negative MEK1 enhanced drug-induced DNA damage whereas expression of activated MEK1 suppressed both the DNA damage response and tumor cell killing. Collectively our data demonstrate that PARP1 and CHK1 inhibitors interact to kill mammary carcinoma cells and that increased DNA damage is a surrogate marker for the response of cells to this drug combination.

We have further defined mechanism(s) by which the drug OSU-03012 (OSU) kills brain cancer cells. OSU toxicity was enhanced by the HSP90 inhibitor 17-N-Allylamino-17-demethoxygeldanamycin (17AAG) that correlated with reduced expression of ERBB1 and ERBB2. Inhibition of the extrinsic apoptosis pathway blocked the interaction between 17AAG and OSU. OSU toxicity was enhanced by the inhibitor of ERBB1/2/4, lapatinib. Knock down of ERBB1/2/4 in a cell line specific fashion promoted OSU toxicity. Combined exposure of cells to lapatinib and OSU resulted in reduced AKT and ERK1/2 activity; expression of activated forms of AKT and to a lesser extent MEK1 protected cells from the lethal effects of the drug combination. Knock down of PTEN suppressed, and expression of PTEN enhanced, the lethal interaction between OSU and lapatinib. Downstream of PTEN, inhibition of mTOR recapitulated the effects of lapatinib. Knock down of CD95, NOXA, PUMA, BIK or AIF, suppressed lapatinib and OSU toxicity. Knock down of MCL-1 enhanced, and overexpression of MCL-1 suppressed, drug combination lethality. Lapatinib and OSU interacted in vivo to suppress the growth of established tumors. Collectively our data argue that the inhibition of ERBB receptor function represents a useful way to enhance OSU lethality in brain tumor cells.

The present studies sought to further understand how the anti-folate pemetrexed and the multi-kinase inhibitor sorafenib interact to kill tumor cells. Sorafenib activated SRC, and via SRC the drug combination activated ERK1/2. Expression of dominant negative SRC or dominant negative MEK1 abolished drug-induced ERK1/2 activation, together with drug-induced autophagy, acidic lysosome formation, and tumor cell killing. Protein phosphatase 2A is an important regulator of the ERK1/2 pathway. Fulvestrant resistant MCF7 cells expressed higher levels of the PP2A inhibitor SET/I2PP2A, had lower endogenous PP2A activity, and had elevated basal ERK1/2 activity compared with their estrogen dependent counterparts. Overexpression of I2PP2A blocked drug-induced activation of ERK1/2 and tumor cell killing. PP2A can be directly activated by ceramide and SET/I2PP2A can be inhibited by ceramide. Inhibition of the de novo ceramide synthase pathway blocked drug-induced ceramide generation, PP2A activation and tumor cell killing. Collectively these findings demonstrate that ERK1/2 plays an essential role downstream of SRC in pemetrexed and sorafenib lethality and that PP2A plays an important role in regulating this process.

The present studies were designed to determine whether the multi-kinase inhibitor sorafenib (Nexavar) interacted with histone deacetylase inhibitors to kill glioblastoma and medulloblastoma cells. In a dose-dependent fashion sorafenib lethality was enhanced in multiple genetically disparate primary human glioblastoma isolates by the HDAC inhibitor sodium valproate (Depakote). Drug exposure reduced phosphorylation of p70 S6K and of mTOR. Similar data to that with valproate were also obtained using the HDAC inhibitor vorinostat (Zolinza). Sorafenib and valproate also interacted to kill medulloblastoma and PNET cell lines. Treatment with sorafenib and HDAC inhibitors radio-sensitized both GBM and medulloblastoma cell lines. Knock down of death receptor (CD95) expression protected GBM cells from the drug combination, as did overexpression of c-FLIP-s, BCL-XL and dominant negative caspase 9. Knock down of PDGFRα recapitulated the effect of sorafenib in combination with HDAC inhibitors. Collectively, our data demonstrate that the combination of sorafenib and HDAC inhibitors kills through activation of the extrinsic pathway, and could represent a useful approach to treat CNS-derived tumors.

The present studies were initiated to determine whether inhibitors of MEK1/2 or SRC signaling, respectively, enhance CHK1 inhibitor lethality in primary human glioblastoma cells. Multiple MEK1/2 inhibitors (CI-1040 (PD184352); AZD6244 (ARRY-142886)) interacted with multiple CHK1 inhibitors (UCN-01, AZD7762) to kill multiple primary human glioma cell isolates that have a diverse set of genetic alterations typically found in the disease. Inhibition of SRC family proteins also enhanced CHK1 inhibitor lethality. Combined treatment of glioma cells with (MEK1/2 + CHK1) inhibitors enhanced radiosensitivity. Combined (MEK1/2 + CHK1) inhibitor treatment led to dephosphorylation of ERK1/2 and S6 ribosomal protein, whereas the phosphorylation of JNK and p38 was increased. MEK1/2 + CHK1 inhibitor-stimulated cell death was associated with the cleavage of pro-caspases 3 and 7 as well as the caspase substrate (PARP). We also observed activation of pro-apoptotic BCL-2 effector proteins BAK and BAX and reduced levels of pro-survival BCL-2 family protein BCL-XL. Overexpression of BCL-XL alleviated but did not completely abolish MEK1/2 + CHK1 inhibitor cytotoxicity in GBM cells. These findings argue that multiple inhibitors of the SRC-MEK pathway have the potential to interact with multiple CHK1 inhibitors to kill glioma cells.

We have further defined mechanism(s) by which the drug OSU-03012 (OSU) kills tumor cells. OSU lethality was suppressed by knock down of PERK and enhanced by knock down of ATF6 and IRE1α. OSU treatment suppressed expression of the chaperone, BiP/GRP78, and did so through reduced stability of the protein. Knock down of BiP/GRP78 further enhanced OSU lethality. Overexpression of BiP/GRP78 abolished OSU toxicity. Pre-treatment of cells with OSU enhanced radiosensitivity to a greater extent than concomitant or sequential drug treatment with radiation exposure. Expression of a mutant active p110 PI3K, or mutant active forms of the EGFR in GBM cells did not differentially suppress OSU killing. In contrast loss of PTEN function reduced OSU lethality, without altering AKT, p70 S6K or mTOR activity, or the drug's ability to radiosensitize GBM cells. Knock down of PTEN protected cells from OSU and radiation treatment whereas re-expression of PTEN facilitated drug lethality and radiosensitization. In a dose-dependent fashion OSU prolonged the survival of mice carrying GBM tumors and interacted with radiotherapy to further prolong survival. Collectively, our data show that reduced BiP/GRP78 levels play a key role in OSU-3012 toxicity in GBM cells, and that this drug has in vivo activity against an invasive primary human GBM isolate.

Pemetrexed (ALIMTA) is a folate anti-metabolite that has been approved for the treatment of non-small cell lung cancer, and has been shown to stimulate autophagy. In the present study, we sought to further understand the role of autophagy in the response to pemetrexed and to test if combination therapy could enhance the level of toxicity through altered autophagy in tumor cells. The multikinase inhibitor sorafenib (NEXAVAR), used in the treatment of renal and hepatocellular carcinoma, suppresses tumor angiogenesis and promotes autophagy in tumor cells. We found that sorafenib interacted in a greater than additive fashion with pemetrexed to increase autophagy and to kill a diverse array of tumor cell types. Tumor cell types that displayed high levels of cell killing after combination treatment showed elevated levels of AKT, p70 S6K and/or phosphorylated mTOR, in addition to class III RTKs such as PDGFRb and VEGFR1, known in vivo targets of sorafenib. In xenograft and in syngeneic animal models of mammary carcinoma and glioblastoma, the combination of sorafenib and pemetrexed suppressed tumor growth without deleterious effects on normal tissues or animal body mass. Taken together, the data suggest that premexetred and sorafenib act synergistically to enhance tumor killing via the promotion of a toxic form of autophagy that leads to activation of the intrinsic apoptosis pathway, and predict that combination treatment represents a future therapeutic option in the treatment of solid tumors.

Introduction: Human cancers are genetically and epigenetically heterogeneous and have the capacity to commandeer a variety of cellular processes to aid in their survival, growth and resistance to therapy. One strategy is to overexpress proteins that suppress apoptosis, such as the Bcl-2 family protein Mcl-1. The Mcl-1 protein plays a pivotal role in protecting cells from apoptosis and is overexpressed in a variety of human cancers., Areas Covered: Targeting Mcl-1 for extinction in these cancers, using genetic and pharmacological approaches, represents a potentially effectual means of developing new efficacious cancer therapeutics. Here we review the multiple strategies that have been employed in targeting this fundamental protein, as well as the significant potential these targeting agents provide in not only suppressing cancer growth, but also in reversing resistance to conventional cancer treatments., Expert Opinion: We discuss the potential issues that arise in targeting Mcl-1 and other Bcl-2 anti-apoptotic proteins, as well problems with acquired resistance. The application of combinatorial approaches that involve inhibiting Mcl-1 and manipulation of additional signaling pathways to enhance therapeutic outcomes is also highlighted. The ability to specifically inhibit key genetic/epigenetic elements and biochemical pathways that maintain the tumor state represent a viable approach for developing rationally based, effective cancer therapies.

The present studies were initiated to determine in greater molecular detail the regulation of CHK1 inhibitor lethality in transfected and infected breast cancer cells and using genetic models of transformed fibrobalsts. Multiple MEK1/2 inhibitors (PD184352, AZD6244 (ARRY-142886)) interacted with multiple CHK1 inhibitors (UCN-01 (7-hydroxystaurosporine), AZD7762) to kill mammary carcinoma cells and transformed fibroblasts. In transformed cells, CHK1 inhibitor -induced activation of ERK1/2 was dependent upon activation of SRC family non-receptor tyrosine kinases as judged by use of multiple SRC kinase inhibitors (PP2, Dasatinib; AZD0530), use of SRC/FYN/YES deleted transformed fibroblasts or by expression of dominant negative SRC. Cell killing by SRC family kinase inhibitors and CHK1 inhibitors was abolished in BAX/BAK -/- transformed fibroblasts and suppressed by over expression of BCL-XL. Treatment of cells with BCL-2/BCL-XL antagonists promoted SRC inhibitor + CHK1 inhibitor -induced lethality in a BAX/BAK-dependent fashion. Treatment of cells with [SRC + CHK1] inhibitors radio-sensitized tumor cells. These findings argue that multiple inhibitors of the SRC-RAS-MEK pathway interact with multiple CHK1 inhibitors to kill transformed cells.

The present studies focused on determining whether the autophagy-inducing drug OSU-03012 (AR-12) could enhance the toxicity of recombinant adenoviral delivery of melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24) in glioblastoma multiforme (GBM) cells. The toxicity of a recombinant adenovirus to express MDA-7/IL-24 (Ad.mda-7) was enhanced by OSU-03012 in a diverse panel of primary human GBM cells. The enhanced toxicity correlated with reduced ERK1/2 phosphorylation and expression of MCL-1 and BCL-XL, and was blocked by molecular activation of ERK1/2 and by inhibition of the intrinsic, but not the extrinsic, apoptosis pathway.  Both OSU-03012 and expression of MDA-7/IL-24 increased phosphorylation of PKR-like endoplasmic reticulum kinase (PERK) that correlated with increased levels of autophagy and expression of dominant negative PERK blocked autophagy induction and tumor cell death. Knockdown of ATG5 or Beclin1 suppressed OSU-03012 enhanced MDA-7/IL-24-induced autophagy and blocked the lethal interaction between the two agents. Ad.mda-7-infected GBM cells secreted MDA-7/IL-24 into the growth media and this conditioned media induced expression of MDA-7/IL-24 in uninfected GBM cells. OSU-03012 interacted with conditioned media to kill GBM cells and knockdown of MDA-7/IL-24 in these cells suppressed tumor cell killing. Collectively, our data demonstrate that the induction of autophagy and mitochondrial dysfunction by a combinatorial treatment approach represents a potentially viable strategy to kill primary human GBM cells.

The Bcl-2 antagonist ABT-737 kills transformed cells in association with displacement of Bim from Bcl-2. The histone deactetylase (HDAC) inhibitor suberoyl bis-hydroxamic acid (SBHA) was employed to determine whether and by what mechanism ABT-737 might interact with agents that upregulate Bim. Expression profiling of BH3-only proteins indicated that SBHA increased Bim, Puma, and Noxa expression, while SBHA concentrations that upregulated Bim significantly potentiated ABT-737 lethality. Concordance between SBHA-mediated Bim upregulation and interactions with ABT-737 was observed in various human leukemia and myeloma cells. SBHA-induced Bim was largely sequestered by Bcl-2 and Bcl-x(L), rather than Mcl-1; ABT-737 attenuated these interactions, thereby triggering Bak/Bax activation and mitochondrial outer membrane permeabilization. Knockdown of Bim (but not Puma or Noxa) by shRNA or ectopic overexpression of Bcl-2, Bcl-x(L), or Mcl-1 diminished Bax/Bak activation and apoptosis. Notably, ectopic expression of these antiapoptotic proteins disabled death signaling by sequestering different proapoptotic proteins, i.e., Bim by Bcl-2, both Bim and Bak by Bcl-x(L), and Bak by Mcl-1. Together, these findings indicate that HDAC inhibitor-inducible Bim is primarily neutralized by Bcl-2 and Bcl-x(L), thus providing a mechanistic framework by which Bcl-2 antagonists potentiate the lethality of agents, such as HDAC inhibitors, which upregulate Bim.

Prior studies demonstrated that resistance to the ERBB1/2 inhibitor Lapatinib in HCT116 cells was mediated by increased MCL-1 expression. We examined whether inhibition of BCL-2 family function could restore Lapatinib toxicity in Lapatinib adapted tumor cells and enhance Lapatinib toxicity in naive cells. The BCL-2 family antagonist Obatoclax (GX15-070), that inhibits BCL-2/BCL-X(L)/MCL-1 function, enhanced Lapatinib toxicity in parental HCT116 and Lapatinib adapted HCT116 cells. In breast cancer lines, regardless of elevated ERBB1/2 expression, GX15-070 enhanced Lapatinib toxicity within 3-12 h. The promotion of Lapatinib toxicity neither correlated with cleavage of caspase 3 nor was blocked by inhibition caspases; and was not associated with changes in the activities of ERK1/2, JNK1/2 or p38 MAPK but with reduced AKT, mTOR and S6K1 phosphorylation. The promotion of Lapatinib toxicity by GX15-070 correlated with increased cytosolic levels of apoptosis inducing factor (AIF) and expression of ATG8 (LC3), and the formation of large vesicles that intensely stained for a transfected LC3-GFP construct. Knock down of the autophagy regulatory proteins ATG5 or Beclin1 suppressed the induction of LC3-GFP vesicularization and significantly reduced cell killing, whereas knock down of MCL-1 and BCL-X(L) enhanced the induction of LC3-GFP vesicularization and significantly enhanced cell killing. Knockdown of Beclin1 and AIF abolished cell killing. Collectively, our data demonstrate that Obatoclax mediated inhibition of MCL-1 rapidly enhances Lapatinib toxicity in tumor cells via a toxic form of autophagy and via AIF release from the mitochondrion.

Mechanisms underlying interactions between the proteasome inhibitor bortezomib and small molecule Bcl-2 antagonists were examined in GC- and ABC-type human DLBCL (diffuse lymphocytic B-cell lymphoma) cells. Concomitant or sequential exposure to non- or minimally toxic concentrations of bortezomib or other proteasome inhibitors and either HA14-1 or gossypol resulted in a striking increase in Bax/Bak conformational change/translocation, cytochrome c release, caspase activation and synergistic induction of apoptosis in both GC- and ABC-type cells. These events were associated with a sharp increase in activation of the stress kinase JNK and evidence of ER stress induction (e.g., eIF2alpha phosphorylation, activation of caspases-2 and -4, and Grp78 upregulation). Pharmacologic or genetic (e.g., shRNA knockdown) interruption of JNK signaling attenuated HA14-1/bortezomib lethality and ER stress induction. Genetic disruption of the ER stress pathway (e.g., in cells expressing caspase-4 shRNA or DN-eIF2alpha) significantly attenuated lethality. The toxicity of this regimen was independent of ROS generation. Finally, HA14-1 significantly increased bortezomib-mediated JNK activation, ER stress induction, and lethality in bortezomib-resistant cells. Collectively these findings indicate that small molecule Bcl-2 antagonists promote bortezomib-mediated mitochondrial injury and lethality in DLBCL cells in association with enhanced JNK activation and ER stress induction. They also raise the possibility that such a strategy may be effective in different DLBCL sub-types (e.g., GC- or ABC), and in bortezomib-resistant disease.

A subtraction hybridization approach combined with a differentiation therapy model of human melanoma identified melanoma differentiation associated gene-7 (mda-7) as a gene upregulated during induction of terminal differentiation. Based on conserved structure, chromosomal location and cytokine-like properties, mda-7, has now been classified as a member of the expanding interleukin (IL)-10 gene family and designated as mda-7/IL-24. Extensive in vitro and in vivo human tumor xenograft studies confirm that mda-7/IL-24 induces apoptosis specifically in tumor cells without harming normal cells. Unique properties of mda-7/IL-24 action also include potent "bystander antitumor" activity, an ability to exert anti-angiogenic effects, immune modulating ability and a capacity to enhance the sensitivity of tumor cells to radiotherapy, chemotherapy and monoclonal antibody therapy. Very recent studies from our groups further reveal autocrine regulation and chemoprevention facilitating properties of mda-7/IL-24. Based on these remarkable antitumor attributes, mda-7/IL-24 was evaluated by intratumoral injection with a replication incompetent adenovirus expressing this gene (Ad.mda-7; INGN 241) in a phase I clinical trial in patients with metastatic melanomas and other advanced solid cancers. mda-7/IL-24 was well tolerated with significant clinical activity. This review highlights our current understanding of the molecular and biochemical basis of mda-7/IL-24 antitumor properties and highlights its potential as a viable gene-based therapy for a wide spectrum of primary and advanced cancers.

We recently noted that low doses of sorafenib and vorinostat interact in a synergistic fashion to kill carcinoma cells by activating CD95, and this drug combination is entering phase I trials. The present studies mechanistically extended our initial observations. Low doses of sorafenib and vorinostat, but not the individual agents, caused an acidic sphingomyelinase and fumonisin B1-dependent increase in CD95 surface levels and CD95 association with caspase 8. Knock down of CD95 or FADD expression reduced sorafenib/vorinostat lethality. Signaling by CD95 caused PERK activation that was responsible for both promoting caspase 8 association with CD95 and for increased eIF2alpha phosphorylation; suppression of eIF2alpha function abolished drug combination lethality. Cell killing was paralleled by PERK-and eIF2alpha-dependent lowering of c-FLIP-s protein levels and overexpression of c-FLIP-s maintained cell viability. In a CD95-, FADD- and PERK-dependent fashion, sorafenib and vorinostat increased expression of ATG5 that was responsible for enhanced autophagy. Expression of PDGFRbeta and FLT3 were essential for high dose single agent sorafenib treatment to promote autophagy. Suppression of PERK function reduced sorafenib and vorinostat lethality whereas suppression of ATG5 levels elevated sorafenib and vorinostat lethality. Overexpression of c-FLIP-s blocked apoptosis and enhanced drug-induced autophagy. Thus sorafenib and vorinostat promote ceramide-dependent CD95 activation followed by induction of multiple downstream survival regulatory signals: ceramide-CD95-PERK-FADD-pro-caspase 8 (death); ceramide-CD95-PERK-eIF2alpha- downward arrowc-FLIP-s (death); ceramide-CD95-PERK-ATG5-autophagy (survival).

The manuscripts by Park et al. and Zhang et al. were initially planned as studies to understand the regulation of cell survival in transformed cells treated with sorafenib and vorinostat, and in primary hepatocytes treated with a bile acid+MEK1/2 inhibitor. In both cell systems we discovered that the toxicity of sorafenib and vorinostat or bile acid+MEK1/2 inhibitor exposure depended on the generation of ceramide and the ligand-independent activation of the CD95 death receptor, with subsequent activation of pro-caspase 8. We noted, however, in these systems that, in parallel with death receptor-induced activation of the extrinsic pathway, CD95 signaling also promoted increased phosphorylation of PKR-like endoplasmic reticulum kinase (PERK) and eIF2alpha, increased expression of ATG5, and increased processing of LC3 and vesicularization of a GFP-LC3 construct. The knockdown of ATG5 expression blocked GFP-LC3 vesicularization and enhanced cell killing. Thus ceramide-CD95 signaling promoted cell death via activation of pro-caspase 8 and cell survival via autophagy. PERK was shown to signal in a switch-hitting fashion; PERK promoted CD95-DISC formation and an eIF2alpha-dependent reduction in c-FLIP-s levels that were essential for cell killing to proceed, but in parallel it also promoted autophagy that was protective. The death receptor-induced apoptosis and autophagy occur proximal to the receptor rather than the mitochondrion, and the relative flow of death receptor signaling into either pathway may determine cell fate. Finally, death receptor induced apoptosis and autophagy could be potential targets for therapeutic intervention.

Glioblastoma multiforme (GBM) is an invariably fatal malignancy. The lethality of GBM has been linked to the highly invasive nature of GBM cells, their escape from immune cell oversight and their high degree of resistance to multiple established therapeutic modalities. The resistance of GBM cells to undergo death processes has, in part, been associated with mutations of specific oncogenes and altered expression of other signaling molecules that lead to reduced capacities to activate multiple apoptosis pathways as well as altered rates of DNA repair and autophagy in response to cytotoxic drugs and cellular stresses. This review will examine how gene therapeutic approaches have been used in the past and are continuing to be used alongside cell signaling modulators and DNA damaging agents as clinical tools to treat GBM. The concerted use of established and novel signal transduction modulatory agents on GBM survival may have potential to lower the apoptotic threshold and facilitate killing in this lethal malignancy.

Melanoma differentiation associated gene-7/interleukin 24 (mda-7/IL-24) is a cytokine displaying selective apoptosis-inducing activity in tumors, including glioblastoma (GBM), without damaging normal cells. The present studies focused on defining whether an adenovirus expressing MDA-7/IL-24, Ad.mda-7, infused into pre-formed invasive primary human GBM tumors growing in athymic mouse brains altered tumor cell growth and animal survival, and whether Ad.mda-7 radiosensitized GBM cells and enhanced the survival benefit of irradiation. Ad.mda-7 directly radiosensitized glioma cells in vitro in a JNK1-3- and caspase 9-dependent fashion and demonstrated bystander-effect killing and radiosensitization of GBM cells when primary human astrocytes were infected with Ad.mda-7. Infusion of Ad.mda-7 into pre-formed glioma tumors caused a rapid decrease in proliferation and blood vessel density and an increase in cell killing. Irradiation of Ad.mda-7 infected tumors enhanced cell death. Cell killing correlated with pro-caspase 3 cleavage, enhanced phosphorylation of JNK1-3 and reduced phosphorylation of ERK1/2. Ad.mda-7 enhanced the survival of animals implanted with GBM6 and GBM12 tumors, and significantly increased the survival benefit of irradiation in animals bearing GBM12 tumors. Ad.mda-7 toxicity was evident against CD133+ and CD133- GBM cells; upon tumor re-growth approximately 70-100 days after virus infusion, the relative CD133+ level within the tumor was profoundly reduced with lower Ki67 reactivity and increased beta-galactosidase staining. Infusion of Ad.mda-7 into an immune competent rat brain did not cause normal tissue toxicity 1-4 weeks after infusion using T1 and T2 weighted MRI and H&E staining. Our data demonstrate that Ad.mda-7 prolongs the survival of animals bearing GBM tumors and does so through multiple mechanisms including direct tumor cell killing and selection for surviving cells that are more differentiated and potentially displaying a putatively senescent phenotype.

Melanoma differentiation associated gene-7/interleukin 24 (mda-7/IL-24) is a novel cytokine displaying selective apoptosis-inducing activity in transformed cells without harming normal cells. The studies by Yacoub et al. (Mol Cancer Ther 2008; 7:314-29) further defines the mechanism(s) by which a GST-MDA-7 fusion protein inhibits cell survival of primary human glioma cells in vitro. GST-MDA-7 killed glioma cells with diverse genetic characteristics that were dependent on activation of JNK1-3 with subsequent activation of BAX and the induction of mitochondrial dysfunction. Activation of JNK1-3 was dependent upon protein kinase R-like endoplasmic reticulum kinase (PERK) and GST-MDA-7 lethality was suppressed in PERK(-/-) cells. GST-MDA-7 caused PERK-dependent vacuolization of LC3-expressing endosomes whose formation was suppressed by incubation with 3-methyladenine, expression of HSP70 or of BiP/GRP78, or by knockdown of ATG5 or Beclin 1 expression, but not by inhibition of the JNK1-3 pathway. Knockdown of ATG5 or Beclin 1 expression or overexpression of HSP70 reduced GST-MDA-7 lethality. Our data demonstrate that GST-MDA-7 induces an ER stress response that, via the induction of autophagy, is causal in the activation of pro-apoptotic pathways that converge on the mitochondrion and ultimately culminate in decreased glioma cell survival.

The manuscript by Park et al. (Mol. Pharm. 2008; mol.107.042697 / PMID: 18182481) further defines the mechanism(s) by which OSU-03012 (OSU) kills transformed cells. It notes that in PKR-like endoplasmic reticulum kinase null cells (PERK-/-) the lethality of OSU is attenuated. OSU enhances the expression of ATG5 in a PERK-dependent fashion and promotes the ATG5-dependent formation of vesicles containing LC3, followed by a subsequent cleavage of cathepsin B and a cathepsin B-dependent formation of low pH intracellular vesicles; cathepsin B is activated and released into the cytosol, and genetic suppression of cathepsin B or AIF function significantly suppresses cell killing. In parallel, OSU causes PERK-dependent increases in HSP70 expression and decreases in HSP90 and Grp78/BiP expression. Inhibition of HSP70 expression enhances OSU toxicity and over-expression of HSP70 suppresses OSU-induced low pH vesicle formation and lethality. Thus, in this system PERK signaling promotes autophagy, which is causally linked to lysosomal dysfunction, cathepsin activation and cell death. However, in parallel, PERK signaling acts to suppress autophagy and lysosomal dysfunction by increasing the expression of HSP70. These findings may help explain why, in a cell type and stimulus-dependent fashion; autophagy has been noted to act either as a protective or as a toxic signal in cells.

The ability of human chorionic gonadotropin (hCG) to modify prostate carcinoma viability in vitro and in vivo when combined with the HMG CoA reductase inhibitor lovastatin and ionizing radiation was investigated. Treatment of PC-3 cells in vitro with hCG caused a modest increase in numbers of non-viable cells within 96 h. Treatment of cells with hCG followed by exposure to the HMG CoA reductase inhibitor lovastatin suppressed AKT phosphorylation and enhanced the cytotoxic effects of hCG. The cytotoxic effects of hCG were blocked by expression of BCL-(XL) and dominant negative caspase 9. Treatment of mice bearing PC-3 flank tumors with lovastatin and hCG significantly reduced tumor volume within 7 days; this was also reflected in decreased ex vivo colony survival of the cells which correlated with increased cleavage of pro-caspase 3 and reduced Ki67 immuno-reactivity. In vitro, treatment of PC-3 cells with hCG followed by exposure to ionizing radiation enhanced the cytotoxic effects of hCG, that was further enhanced by lovastatin. In vivo, hCG radiosensitized PC-3 tumors and significantly enhanced the lethality of hCG and lovastatin treatment. Collectively, our findings argue that treatment of PC-3 prostate cancer tumors with hCG, lovastatin and radiation represents a potential novel therapeutic approach.

Sorafenib is a multikinase inhibitor that induces apoptosis in human leukemia and other malignant cells. Recently, we demonstrated that sorafenib diminishes Mcl-1 protein expression by inhibiting translation through a MEK1/2-ERK1/2 signaling-independent mechanism and that this phenomenon plays a key functional role in sorafenib-mediated lethality. Here, we report that inducible expression of constitutively active MEK1 fails to protect cells from sorafenib-mediated lethality, indicating that sorafenib-induced cell death is unrelated to MEK1/2-ERK1/2 pathway inactivation. Notably, treatment with sorafenib induced endoplasmic reticulum (ER) stress in human leukemia cells (U937) manifested by immediate cytosolic-calcium mobilization, GADD153 and GADD34 protein induction, PKR-like ER kinase (PERK) and eukaryotic initiation factor 2alpha (eIF2alpha) phosphorylation, XBP1 splicing, and a general reduction in protein synthesis as assessed by [35S]methionine incorporation. These events were accompanied by pronounced generation of reactive oxygen species through a mechanism dependent upon cytosolic-calcium mobilization and a significant decline in GRP78/Bip protein levels. Interestingly, enforced expression of IRE1alpha markedly reduced sorafenib-mediated apoptosis, whereas knockdown of IRE1alpha or XBP1, disruption of PERK activity, or inhibition of eIF2alpha phosphorylation enhanced sorafenib-mediated lethality. Finally, downregulation of caspase-2 or caspase-4 by small interfering RNA significantly diminished apoptosis induced by sorafenib. Together, these findings demonstrate that ER stress represents a central component of a MEK1/2-ERK1/2-independent cell death program triggered by sorafenib.

Taxol (paclitaxel) and Taxotere (docetaxel) are considered as two of the most important anti-cancer chemotherapy drugs. The cytotoxic action of these drugs has been linked to their ability to inhibit microtubule depolymerization, causing growth arrest and subsequent cell death. Studies by a number of laboratories have also linked suppression of MEK1/2 signaling to enhanced Taxol toxicity in vitro and in vivo. The present study examined the interactions of the semi-synthetic taxane Taxotere with MEK1/2 inhibitors in epithelial tumor cells. In vitro colony formation studies demonstrated that Taxotere and the MEK1/2 inhibitor PD184352 interacted in a sequence dependent fashion to synergistically kill human mammary carcinoma cells (MDA-MB-231, MCF7) as well as in other tumor cell types; e.g. prostate and renal cell carcinoma. Athymic mice were implanted in the rear flank with either MDA-MB-231 or MCF7 cells and tumors permitted to form to a volume of approximately 100 mm3 prior to a two day exposure of either Vehicle, PD184352 (25 mg/kg), Taxotere (15 mg/kg) or the drug combination. Tumor volume was measured every other day and tumor growth determined over the following approximately 30 days. Transient exposure of MDA-MB-231 tumors or MCF7 tumors to PD184352 did not significantly alter tumor growth rate or the mean tumor volume in vivo approximately 15-30 days after drug administration. Transient Taxotere exposure of MDA-MB-231 or to a lesser extent MCF7, tumors modestly reduced the mean tumor volume in vivo approximately 15-30 days after drug administration. In contrast, combined treatment with PD184352 and Taxotere significantly reduced MDA-MB-231 and MCF7 tumor growth. The tumor control values for MDA-MB-231 cells and MCF7 cells were 0.43 and 0.71, respectively. Fractionated irradiation of MDA-MB-231 tumors during drug exposure or single dose irradiation prior to drug administration did not significantly further suppress tumor growth beyond that of cells exposed to Taxotere and MEK1/2 inhibitor. Single dose irradiation of tumors after drug exposure, however, caused a significant further suppression of tumor growth below that caused by drug exposure. These findings were also reflected in ex vivo colony formation analyses of isolated tumor cells. Collectively, these findings argue that Taxotere and MEK1/2 inhibitors have the potential to suppress mammary tumor growth in vivo which is enhanced by sequence-dependent exposure to ionizing radiation. Based on the cell lines used in these studies, our findings argue that the interaction of Taxotere and PD184352 is independent of p53 status, estrogen dependency, caspase 3 levels or oncogenic K-RAS expression.

Dysregulation of cyclin D1 expression is one of the most common genetic aberrations found in hematopoietic malignancies, including multiple myeloma. To address the effects of cyclin D1 overexpression might have on the response of malignant hematopoietic cells to CDK inhibitors, the impact of ectopic cyclin D1 overexpression on the response of human multiple myeloma U266 cells to various cyclin-dependent kinase (CDK) inhibitors was examined. Cyclin D1 overexpression markedly increased the apoptotic response of cells to the CDK inhibitors flavopiridol, roscovitine, and R-roscovitine. Ectopic expression of cyclin D1 resulted in p21(CIP1) accumulation, an effect that was diminished by CDK inhibitor exposure. In pRb-null U266 cells, enforced overexpression of cyclin D1 diminished CDK inhibitor-mediated dephosphorylation of the pocket proteins p130 and p107, reduced binding of E2F1 and E2F4 to p130 and p107, and attenuated inhibition of E2F activity. Notably, CDK inhibitors failed to reduce the S phase fraction in cyclin D1/U266 cells in contrast to effects in their wild-type counterparts. Finally, cyclin D1/U266 cells exhibited diminished basal NF-kappaB activity compared to controls, which was essentially completely abrogated by CDK inhibitor exposure. Together, these findings suggest that dysregulation of cyclin D1 sensitizes human myeloma cells to the actions of CDK inhibitors through mechanisms involving interference with p21(CIP1) expression, dephosphorylation of pocket proteins and inactivation of E2Fs culminating in S phase entry, as well as inactivation of NF-kappaB, leading to apoptosis rather than growth arrest.

Previous studies from our group have demonstrated in vitro that UCN-01 (7-hydroxystaurosporine) and inhibitors of MEK1/2 interact to cause tumor cell death in a wide variety of malignant, but not in nontransformed, cell types. The present studies determined whether UCN-01 and MEK1/2 inhibitors interacted to cause tumor cell death in vivo. In vitro colony formation studies demonstrated that UCN-01 and the MEK1/2 inhibitor PD184352 interacted to synergistically kill human mammary carcinoma cells (MDA-MB-231, MCF7) with similar combination index values. Athymic mice were implanted in the rear flank with either MDA-MB-231 or MCF7 cells and tumors permitted to form to a volume of approximately 100 mm3 prior to a two day exposure of either Vehicle, PD184352 (25 mg/kg), UCN-01 (0.1-0.2 mg/kg) or the drug combination. Tumor volume was measured every other day and tumor growth determined over the following approximately 30 days. Transient exposure of MDA-MB-231 tumors or MCF7 tumors to either PD184352 or UCN-01 did not significantly alter tumor growth rate or the mean tumor volume in vivo approximately 15-30 days after drug administration. In contrast, combined treatment with PD184352 and UCN-01 significantly reduced MDA-MB-231, and largely abolished MCF7 tumor growth. Tumor control values for both cell lines were 0.36. Tumor cells isolated approximately 30 days after combined drug exposure exhibited a significantly greater reduction in plating efficiency using ex vivo colony formation assays than tumor cells that were exposed to either drug individually. Reduced tumor growth correlated with profound tumor cell death within five days of combined drug exposure, which was also evident approximately 30 days after exposure. In addition, tumor cell death correlated with a reduction in the phosphorylation of ERK1/2 and the immuno-reactivity of Ki67 and of CD31. Collectively, these findings argue that UCN-01 and MEK1/2 inhibitors have the potential to suppress mammary tumor growth in vivo which is independent of p53 status, estrogen dependency, caspase 3 levels or oncogenic K-RAS expression.