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.
medulloblastoma; PDE5; chemotherapy; sildenafil; pediatric
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.
autophagy; sarcoma; CD95
Despite modest improvements in survival over the last several decades, the treatment of AML continues to present a formidable challenge. Most patients are elderly, and these individuals, as well as those with secondary, therapy-related, or relapsed/refractory AML, are particularly difficult to treat, owing to both aggressive disease biology and the high toxicity of current chemotherapeutic regimens. It has become increasingly apparent in recent years that coordinated interruption of cooperative survival signaling pathways in malignant cells is necessary for optimal therapeutic results. The modest efficacy of monotherapy with both cytotoxic and targeted agents in AML testifies to this. As the complex biology of AML continues to be elucidated, many “synthetic lethal” strategies involving rational combinations of targeted agents have been developed. Unfortunately, relatively few of these have been tested clinically, although there is growing interest in this area. In this article, the preclinical and, where available, clinical data on some of the most promising rational combinations of targeted agents in AML are summarized. While new molecules should continue to be combined with conventional genotoxic drugs of proven efficacy, there is perhaps a need to rethink traditional philosophies of clinical trial development and regulatory approval with a focus on mechanism-based, synergistic strategies.
AML; targeted therapies; rational combinations; HDAC inhibitors; CDK inhibitors; proteasome inhibitors; checkpoint abrogators; apoptosis; BH3-mimetics; Mcl-1
Prostate cancer is the second leading cause of cancer-related deaths in men in the U.S. At present, no single or combination therapy has shown efficacy in decreasing disease progression in patients with metastatic disease. A potentially viable approach for treating late-stage prostate cancer is gene therapy. Adenoviruses (Ad) are the most commonly used mode of gene delivery, but progress using this vector has been hampered by concerns over the safety and practicality of viruses including conditionally replicating Ads (CRAds), particularly for intravenous delivery, and the inefficiency of non-viral transfection techniques. Major challenges for effective gene therapy using Ads are the limited infectivity of regular Ad serotype 5 (Ad5) and the inability to specifically deliver the therapeutic directly into diseased tissue without trapping in the liver or elimination by the immune system. The shortcoming in using Ad5 is mostly attributed to a reduction in Coxsackie-adenovirus receptors (CAR) on the surface of cancer cells, which can be mitigated by generating tropism-modified Ads permitting CAR-independent infection of tumor cells. The limitations of systemic gene delivery can now be overcome by using a novel targeted-delivery approach such as ultrasound (US) contrast agents (microbubbles) to deliver effective therapeutic reagents, Ads, or recombinant proteins, combined with ultrasound-targeted microbubble destruction (UTMD), to develop a site-specific therapy in immune competent transgenic mouse models. These unique strategies for enhancing the efficacy of gene therapy provide a direct path to translation from the laboratory into the clinic for developing an effective gene therapy of prostate cancer.
Evidence implicating dysregulation of the IRE1/XBP-1s arm of the unfolded protein response (UPR) in cancer pathogenesis (e.g., multiple myeloma) has prompted the development of IRE1 RNase inhibitors. Here, effects of cyclin-dependent kinase inhibitor, SCH727965 (dinaciclib), on the IRE1 arm of the UPR were examined in human leukemia and myeloma cells. Exposure of cells to extremely low (e.g., nM) concentrations of SCH727965, a potent inhibitor of CDKs 1/2/5/9, diminished XBP-1s and Grp78 induction by the ER stress-inducers thapsigargin (Tg) and tunicamycin (Tm), while sharply inducing cell death. SCH727965, in contrast to IRE1 RNase inhibitors, inhibited the UPR in association with attenuation of XBP-1s nuclear localization and accumulation rather than transcription, translation, or XBP-1 splicing. Notably, in human leukemia cells, CDK1 and CDK5 shRNA knock-down diminished Grp78 and XBP-1s up-regulation while increasing Tg lethality, arguing for a functional role for CDK1/5 in activation of the cytoprotective IRE1/XBP-1s arm of the UPR. In contrast, CDK9 or CDK2 inhibitors or shRNA knockdown failed to down-regulate XBP-1s or Grp78. Furthermore, IRE1, XBP-1, or Grp78 knockdown significantly increased Tg lethality, as observed with CDK1/5 inhibition/knockdown. Finally, SCH727965 diminished myeloma cell growth in vivo in association with XBP-1s down-regulation. Together, these findings demonstrate that SCH727965 acts at extremely low concentrations to attenuate XBP-1s nuclear accumulation and Grp78 up-regulation in response to ER stress inducers. They also highlight a link between specific components of the cell cycle regulatory apparatus (e.g., CDK1/5) and the cytoprotective IRE1/XBP-1s/Grp78 arm of the UPR that may be exploited therapeutically in UPR-driven malignancies.
SCH727965 (dinaciclib); X-box–binding protein-1 (XBP-1); Grp78/BiP; unfolded protein response
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.
AIF; HDAC inhibitor; RIP-1; autophagy; glioblastoma; mTOR; necro-apoptosis; salinomycin; stem cell
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.
The present studies were designed to compare and contrast the abilities of TRAIL (death receptor agonist) and obatoclax (BCL-2 family inhibitor) to enhance [sorafenib + HDAC inhibitor] toxicity in GI tumor cells. Sorafenib and HDAC inhibitor treatment required expression of CD95 to kill GI tumor cells in vitro and in vivo. In cells lacking CD95 expression, TRAIL treatment, and to a lesser extent obatoclax, enhanced the lethal effects of [sorafenib + HDAC inhibitor] exposure. In hepatoma cells expressing CD95 a similar data pattern emerged with respect to the actions of TRAIL. Downstream of the death receptor the ability of TRAIL to enhance cell killing correlated with reduced AKT, ERK1/2, p70 S6K and mTOR activity and enhanced cleavage of pro-caspase 3 and reduced expression of MCL-1 and BCL-XL. Over-expression of BCL-XL or MCL-1 or expression of dominant negative pro-caspase 9 protected cells from drug toxicity. Expression of activated AKT, p70 S6K, mTORand to a lesser extent MEK1EE also protected cells that correlated with maintained c-FLIP-s expression, reduced BIM expression and increased BAD phosphorylation. In vivo [sorafenib + HDAC inhibitor] toxicity against tumors was increased in a greater than additive fashion by TRAIL.Collectively, our data argue that TRAIL, rather than obatoclax, is the most efficacious agent at promoting [sorafenib + HDAC inhibitor] lethality.
sorafenib; histone deacetylase inhibitor; kinase; apoptosis; CD95; TRAIL; MCL-1
Intracellular proteolytic pathways have been validated as rational targets in multiple myeloma with the approval of two proteasome inhibitors in this disease, and with the finding that immunomodulatory agents work through an E3 ubiquitin ligase containing Cereblon. Another E3 ligase that could be a rational target is the murine double minute (MDM) 2 protein, which plays a role in p53 turnover. A novel inhibitor of this complex, MI-63, was found to induce apoptosis in p53 wild-type myeloma models in association with activation of a p53-mediated cell death program. MI-63 overcame adhesion-mediated drug resistance, showed anti-tumor activity in vivo, enhanced the activity of bortezomib and lenalidomide, and also overcame lenalidomide resistance. In mutant p53 models, inhibition of MDM2 with MI-63 also activated apoptosis, albeit at higher concentrations, and this was associated with activation of autophagy. When MI-63 was combined with the BH3 mimetic ABT-737, enhanced activity was seen in both wild-type and mutant p53 models. Finally, this regimen showed efficacy against primary plasma cells from patients with newly diagnosed and relapsed/refractory myeloma. These findings support the translation of novel MDM2 inhibitors both alone, and in combination with other novel agents, to the clinic for patients with multiple myeloma.
Interactions between the novel Chk1 inhibitor MK-8776 and the HDAC inhibitor (HDACI) vorinostat were examined in human leukemia cells harboring wild-type (wt) or deficient p53. MK-8776 synergistically potentiated vorinostat-mediated apoptosis in various p53-wild type (wt) or -deficient leukemia cell lines, while p53 knock-down by shRNA sensitized p53-wt cells to lethality of this regimen. Leukemia cell lines carrying FLT3-ITD were also sensitive to the MK-8776/vorinostat regimen. Synergistic interactions were associated with inhibition of Chk1 activity, interference with the intra-S phase checkpoint, disruption of DNA replication, and down-regulation of proteins involved in DNA replication (e.g.,CDT1) and repair (e.g., CtIP and BRCA1), resulting in sharp increases in DNA damage, reflected by enhanced γH2A.X formation, and apoptosis. Moreover, leukemia cells expressing kinase-dead Chk1 (D130A) or Chk1 shRNA were significantly more sensitive to HDACIs compared to their wild-type counterparts, and displayed down-regulation of CtIP and BRCA1 phosphorylation following HDACI exposure. Finally, the MK-8776/vorinostat regimen was active in primary AML blasts, particularly against the CD34+/CD38-/CD123+ population enriched for leukemia-initiating cells. In contrast, identical regimens were relatively sparing toward normal cord blood CD34+ cells. Together, these findings indicate that the novel Chk1 inhibitor MK-8776 markedly potentiates HDACI lethality in leukemia cells displaying various genetic backgrounds through mechanisms involving disruption of the intra-S checkpoint, DNA replication, and DNA repair. They also argue that leukemic cells, including those bearing oncogenic mutations associated with poor prognosis e.g., p53 deletion/mutation or FLT3-ITD, may also be susceptible to this strategy.
Chk1 inhibitor; HDAC inhibitor; S phase; DNA damage/repair; leukemia
Cyclin-dependent kinases (CDKs) regulate cell cycle progression.
Certain CDKs (e.g., CDK7, CDK9) also control cellular transcription.
Consequently, CDKs represent attractive targets for anti-cancer 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.
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.
In general, the anti-tumor 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.
PARP1; CHK1; DNA damage; ATM; kinase; apoptosis; comet
This phase I study was conducted to identify the MTD of alvocidib when combined vorinostat in patients with relapsed, refractory, or poor prognosis acute leukemia, or refractory anemia with excess blasts-2 (RAEB-2). Secondary objectives included investigating the pharmacokinetic and pharmacodynamic effects of the combination.
Patients received vorinostat (200 mg orally, 3 times a day [TID], for 14 days), on a 21-day cycle, combined with 2 different alvocidib administration schedules: a 1-h intravenous infusion, daily x 5; or a 30-min loading infusion followed by a 4-h maintenance infusion, weekly x 2. The alvocidib dose was escalated using a standard 3+3 design.
Twenty-eight patients were enrolled and treated. The alvocidib MTD was 20 mg/m2 (30-min loading infusion) followed by 20 mg/m2 (4-h maintenance infusion) on days 1 and 8, in combination with vorinostat. The most frequently encountered toxicities were cytopenias, fatigue, hyperglycemia, hypokalemia, hypophosphatemia, and QT prolongation. Dose limiting toxicities (DLTs) were cardiac arrhythmia-atrial fibrillation and QT prolongation. No objective responses were achieved, although 13 of 26 evaluable patients exhibited stable disease. Alvocidib appeared to alter vorinostat pharmacokinetics, whereas alvocidib pharmacokinetics were unaffected by vorinostat. Ex vivo exposure of leukemia cells to plasma obtained from patients after alvocidib treatment blocked vorinostat-mediated p21CIP1 induction and down-regulated Mcl-1 and p-RNA Pol II for some specimens, although parallel in vivo bone marrow responses were infrequent.
Alvocidib combined with vorinostat is well tolerated. Although disease stabilization occurred in some heavily pretreated patients, objective responses were not obtained with these schedules.
Vorinostat; Alvocidib; Acute Leukemia; Clinical Trial; Phase I
Interactions between the Bruton tyrosine kinase (BTK) inhibitor PCI-32765 and the proteasome inhibitor (bortezomib) were examined in diffuse large-B cell lymphoma (DLBCL) and mantle cell lymphoma (MCL) cells, including those highly resistant to bortezomib. Co-administration of PCI-32765/bortezomib synergistically increased mitochondrial injury and apoptosis in germinal centre- or activated B-cell-like-DLBCL cells and in MCL cells. These events were accompanied by marked AKT and nuclear factor (NF)-κB (NFKB1) inactivation, down-regulation of Mcl-1 (MCL1), Bcl-xL (BCL2L1), and XIAP, and enhanced DNA damage (e.g., γH2A.X formation) and endoplasmic reticulum (ER) stress. Similar interactions were observed in highly bortezomib-resistant DLBCL and MCL cells, and in primary DLBCL cells. In contrast, PCI-32765/bortezomib regimens displayed minimal toxicity toward normal CD34+ bone marrow cells. Transfection of DLBCL cells with a constitutively active AKT construct attenuated AKT inactivation and significantly diminished cell death, whereas expression of an NF-κB “super-repressor” (IκBαser34/36) increased both PCI-32765 and bortezomib lethality. Moreover, cells in which the ER stress response was disabled by a dominant-negative eIF2α construct were resistant to this regimen. Finally, combined exposure to PCI-32765 and bortezomib resulted in more pronounced and sustained reactive oxygen species (ROS) generation, and ROS scavengers significantly diminished lethality. Given promising early clinical results for PCI-32765 in DLBCL and MCL, a strategy combining BTK/ proteasome inhibitor warrants attention in these malignancies.
PCI32765; bortezomib; BTK; DLBCL; mantle cell lymphoma
The anti-apoptotic protein Mcl-1 plays a major role in multiple myeloma (MM) cell survival as well as bortezomib- and microenvironmental forms of drug resistance in this disease. Consequently, there is a critical need for strategies capable of targeting Mcl-1-dependent drug resistance in MM. The present results indicate that a regimen combining Chk1 with MEK1/2 inhibitors effectively kills cells displaying multiple forms of drug resistance stemming from Mcl-1 up-regulation in association with direct transcriptional Mcl-1 down-regulation and indirect disabling of Mcl-1 anti-apoptotic function through Bim up-regulation and increased Bim/Mcl-1 binding. These actions release Bak from Mcl-1, accompanied by Bak/Bax activation. Analogous events were observed in both drug-naïve and acquired bortezomib-resistant MM cells displaying increased Mcl-1 but diminished Bim expression, or cells ectopically expressing Mcl-1. Moreover, concomitant Chk1 and MEK1/2 inhibition blocked Mcl-1 up-regulation induced by IL-6/IGF-1 or co-culture with stromal cells, effectively overcoming microenvironment-related drug resistance. Finally, this regimen down-regulated Mcl-1 and robustly killed primary CD138+ MM cells, but not normal hematopoietic cells. Together, these findings provide novel evidence that this targeted combination strategy could be effective in the setting of multiple forms of Mcl-1-related drug resistance in MM.
Effects of concomitant inhibition of the PI3K/AKT/mTOR pathway and Bcl-2/Bcl-xL (BCL2L1) were examined in human myeloid leukemia cells. Tetracycline-inducible Bcl-2 and Bcl-xL dual knockdown sharply increased PI3K/AKT/mTOR inhibitor lethality. Conversely, inducible knockdown or dominant-negative AKT increased whereas constitutively active AKT reduced lethality of the Bcl-2/Bcl-xL inhibitor ABT-737. Furthermore, PI3K/mTOR inhibitors (e.g., BEZ235, PI-103) synergistically increased ABT-737-mediated cell death in multiple leukemia cell lines and reduced colony-formation in leukemic but not normal CD34+ cells. Notably, increased lethality was observed in 4/6 primary AML specimens. Responding, but not non-responding, samples exhibited basal AKT phosphorylation. PI3K/mTOR inhibitors markedly down-regulated Mcl-1 but increased Bim binding to Bcl-2/Bcl-xL; the latter effect was abrogated by ABT-737. Combined treatment also markedly diminished Bax/Bak binding to Mcl-1, Bcl-2 or Bcl-xL. Bax, Bak, or Bim (BCL2L11) knockdown, or Mcl-1 over-expression significantly diminished regimen-induced apoptosis. Interestingly, pharmacologic inhibition or shRNA knockdown of GSK3α/β significantly attenuated Mcl-1 down-regulation and decreased apoptosis. In a systemic AML xenograft model, dual tet-inducible knockdown of Bcl-2/Bcl-xL sharply increased BEZ235 anti-leukemic effects. In a subcutaneous xenograft model, BEZ235 and ABT-737 co-administration significantly diminished tumor growth, down-regulated Mcl-1, activated caspases, and prolonged survival. Together, these findings suggest that anti-leukemic synergism between PI3K/AKT/mTOR inhibitors and BH3 mimetics involves multiple mechanisms, including Mcl-1 down-regulation, release of Bim from Bcl-2/Bcl-xL as well as Bak and Bax from Mcl-1/Bcl-2/Bcl-xL, and GSK3α/β, culminating in Bax/Bak activation and apoptosis. They also argue that combining PI3K/AKT/mTOR inhibitors with BH3-mimetics warrants attention in AML, particularly in the setting of basal AKT activation and/or addiction.
PI3K; Bcl-2; Bcl-xL; apoptosis; leukemia
To determine whether PLK1 inhibitors (e.g. BI2536) and HDAC inhibitor (e.g. vorinostat) interact synergistically in CML cells sensitive or resistant to imatinib mesylate (IM) in vitro and in vivo.
K562 and LAMA84 cells sensitive or resistant to IM and primary CML cells were exposed to BI2536 and vorinostat. Effects on cell viability and signaling pathways were determined using flow cytometry, western blotting, and gene transfection. K562 and BV173/E255K animal models were used to test in vivo efficacy.
Co-treatment with BI2536 and vorinostat synergistically induced cell death in parental or IM-resistant BCR/ABL+ cells and and primary CD34+ bone marrow cells but was minimally toxic to normal cells. BI2536/vorinostat co-treatment triggered pronounced mitochondrial dysfunction, inhibition of p-BCR/ABL, caspase activation, PARP cleavage, ROS generation, and DNA damage (manifest by increased expression of γH2A.X, p-ATM, p-ATR), events attenuated by the anti-oxidant TBAP. PLK1 shRNA knockdown significantly increased HDACI lethality, whereas or HDAC 1–3 shRNA knockdown reciprocally increased BI2536-induced apoptosis. Genetic interruption of the DNA damage linker H1.2 partially but significantly reduced PLK1/HDAC inhibitor-mediated cell death, suggesting a functional role for DNA damage in lethality. Finally, BI2536/vorinostat co-treatment dramatically reduced tumor growth in both subcutaneous and systemic BCR/ABL+ leukemia xenograft models and significantly enhanced animal survival.
These findings suggest that concomitant PLK1 and HDAC inhibition is active against IM-sensitive or refractory CML cells both in vitro and in vivo, and that this strategy warrants further evaluation in the setting of BCR/ABL+ leukemias.
BI2536; vorinostat; CML; PLK-1; BCR/ABL+
Melanoma differentiation associated gene-9 (mda-9/syntenin) encodes an adapter scaffold protein whose expression correlates with and mediates melanoma progression and metastasis. Tumor angiogenesis represents an integral component of cancer metastasis prompting us to investigate a possible role of mda-9/syntenin in inducing angiogenesis. Genetic (gain-of-function and loss-of-function) and pharmacological approaches were employed to modify mda-9/syntenin expression in normal immortal melanocytes, early radial growth phase melanoma and metastatic melanoma cells. The consequence of modifying mda-9/syntenin expression on angiogenesis was evaluated using both in vitro and in vivo assays, including tube formation assays using human vascular endothelial cells, CAM assays and xenograft tumor animal models. Gain-of-function and loss-of-function experiments confirm that MDA-9/syntenin induces angiogenesis by augmenting expression of several pro-angiogenic factors/genes. Experimental evidence is provided for a model of angiogenesis induction by MDA-9/syntenin in which MDA-9/syntenin interacts with the ECM activating Src and FAK resulting in activation by phosphorylation of Akt, which induces HIF-1α. The HIF-1α activates transcription of Insulin Growth Factor Binding Protein-2 (IGFBP-2), which is secreted thereby promoting angiogenesis and further induces endothelial cells to produce and secrete VEGF-A augmenting tumor angiogenesis. Our studies delineate an unanticipated cell non-autonomous function of MDA-9/syntenin in the context of angiogenesis, which may directly contribute to its metastasis-promoting properties. As a result, targeting MDA-9/syntenin or its downstream-regulated molecules may provide a means of simultaneously impeding metastasis by both directly inhibiting tumor cell transformed properties (autonomous) and indirectly by blocking angiogenesis (non-autonomous).
mda-9/syntenin; melanoma; angiogenesis; IGFBP-2; HuVECs; CAM assay
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-Xl/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. Knockdown 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-Xl 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.
lapatinib; obatoclax; autophagy; cell death; resistance
Despite the remarkable success of imatinib against Bcr-Abl, development of secondary resistance, most often due to point mutations in the Bcr-Abl tyrosine kinase (TK) domain, is quite common. Of these, the T315I “gatekeeper” mutation is resistant to all currently registered Bcr-Abl TK inhibitors (TKIs) with the notable exception of ponatinib (Iclusig™), which was very recently approved by the United States Food and Drug Administration (FDA). Besides ponatinib, numerous strategies have been developed to circumvent this problem. These include the protein synthesis inhibitor omacetaxine (Synribo®), and “switch-control” inhibitors. Dual Bcr-Abl and aurora kinase inhibitors represent another promising strategy. Finally, several promising synergistic combinations, such as TKIs with histone deacetylase inhibitors (HDACIs), warrant attention.
Gatekeeper mutations; T315I; Bcr-Abl; ponatinib; homoharringtonine; omacetaxine; AT9283; HDAC inhibitors
The B-cell lymphoma-2 (Bcl-2) family of proteins regulates the intrinsic, or mitochondrial pathway of apoptosis, the final common mechanism of cell death in response to a variety of physiologic and pharmacologic signals, and plays a central role in AML pathogenesis, prognosis and responsiveness to chemotherapy. Traditionally thought to be an important survival factor for multiple myeloma cells, the anti-apoptotic Bcl-2 family protein myeloid cell leukemia-1 (Mcl-1) has recently been shown in preclinical studies to be critical to the development and maintenance of AML, making it an attractive therapeutic target in this disease. Several characteristics, such as its very short half-life, distinguish Mcl-1 from other anti-apoptotic Bcl-2 family members. Additionally, Mcl-1 levels are regulated by a large number of pathways affecting its transcription, translation and degradation. A variety of approaches exploiting these features has been developed to inhibit directly or indirectly the anti-apoptotic function of Mcl-1. Many of these lend themselves well to combination therapies, leading to striking synergism, at least in preclinical models. In this brief review, we highlight some of the more promising strategies targeting Mcl-1 in AML, with a particular emphasis on rational combinations of novel agents.
AML; Mcl-1; navitoclax; obatoclax; sorafenib; CDK inhibitors
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.
glioblastoma; medulloblastoma; lapatinib; OSU-03012; apoptosis; autophagy; ERBB1; PTEN
BIM represents a BH3-only proapoptotic member of the BCL-2 family of apoptotic regulatory proteins. Recent evidence suggests that in addition to its involvement in normal homeostasis, BIM plays a critical role in tumor cell biology, including the regulation of tumorigenesis through activities as a tumor suppressor, tumor metastasis, and tumor cell survival. Consequently, BIM has become the focus of intense interest as a potential target for cancer chemotherapy. The control of BIM expression is complex, and involves multiple factors, including epigenetic events (i.e., promoter acetylation or methylation, miRNA), transcription factors, posttranscriptional regulation, and posttranslational modifications, most notably phosphorylation. Significantly, the expression of BIM by tumor cells has been shown to play an important role in determining the response of transformed cells to not only conventional cytotoxic agents, but also to a broad array of targeted agents that interrupt cell signaling and survival pathways. Furthermore, modifications in BIM expression may be exploited to improve the therapeutic activity and potentially the selectivity of such agents. It is likely that evolving insights into the factors that regulate BIM expression will ultimately lead to novel BIM-based therapeutic strategies in the future.
BIM; regulation; BCL-2; apoptosis; transcription; translation
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 Ca2+. 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.
MDA-7/IL-24; HDACI; ceramide; apoptosis; bystander; cytokine; ROS; caspase; animal study