Selective Apoptosis Of Cancer Cells Research Articles

Turn up the cellular power generator with vitamin E analogue formulation.

The down regulation of the cellular power generator, adenosine triphosphate (ATP) synthase, in various cancer cells plays an obstructive role in mitochondria-mediated cell death. Cancer cells up-regulate ATPase inhibitory factor 1 (IF1) and down-regulate β-F1-ATPase of ATP synthase to enhance aerobic glycolysis for tumor growth via inhibiting total ATP synthase activity in the oxidative phosphorylation (OXPHOS) pathway. Alpha-tocopheryl succinate (α-TOS), one of the most bioactive derivatives of vitamin E, can selectively induce apoptosis in numerous cancer cells. The cancer cell selective apoptosis inducing property of α-TOS is correlated to: mitochondrial destabilization, inhibition of anti-apoptotic B cell lymphoma 2 (Bcl2) and protein kinase C (PKC), caspase 3 activation, production of mitochondrial reactive oxygen species (ROS), and inhibition of succinate dehydrogenase activity of mitochondrial complex II, and interaction with complex I to some extent. There is no report which elucidates the effects of α-TOS on the cellular power generator, complex V or ATP synthase. Here, we report the activation of mitochondrial ATP synthase using a suitably designed chemical formulation of α-TOS for the first time. A mitochondria targeted α-TOS nanoparticle formulation demonstrated enhanced cytotoxicity and mitochondrial activities in cancer cells by inhibiting Bcl2 protein and activating ATP synthase. The modulation of ATP synthase in cancer cells by the engineered formulation of α-TOS can be promising for solid cancers with compromised ATP synthase.

β-catenin nuclear translocation in colorectal cancer cells is suppressed by PDE10A inhibition, cGMP elevation, and activation of PKG.

Phosphodiesterase 10A (PDE10) is a cGMP and cAMP degrading PDE isozyme that is highly expressed in the brain striatum where it appears to play an important role in cognition and psychomotor activity. PDE10 inhibitors are being developed for the treatment of schizophrenia and Huntington's disease and are generally well tolerated, possibly because of low expression levels in most peripheral tissues. We recently reported high levels of PDE10 in colon tumors and that genetic silencing of PDE10 by siRNA or inhibition with small molecule inhibitors can suppress colon tumor cell growth with a high degree of selectivity over normal colonocytes (Li et al., Oncogene 2015). These observations suggest PDE10 may have an unrecognized role in tumorigenesis. Here we report that the concentration range by which the highly specific PDE10 inhibitor, Pf-2545920 (MP-10), inhibits colon tumor cell growth parallels the concentration range required to increase cGMP and cAMP levels, and activates PKG and PKA, respectively. Moreover, PDE10 knockdown by shRNA reduces the sensitivity of colon tumor cells to the growth inhibitory activity of Pf-2545920. Pf-2545920 also inhibits the translocation of β-catenin to the nucleus, thereby reducing β-catenin mediated transcription of survivin, resulting in caspase activation and apoptosis. PDE10 mRNA was also found to be elevated in colon tumors compared with normal tissues. These findings suggest that PDE10 can be targeted for cancer therapy or prevention whereby inhibition results in cGMP elevation and PKG activation to reduce β-catenin-mediated transcription of survival proteins leading to the selective apoptosis of cancer cells.

Development of Inhibitors of Protein-protein Interactions through REPLACE: Application to the Design and Development Non-ATP Competitive CDK Inhibitors.

REPLACE is a unique strategy developed to more effectively target protein-protein interactions (PPIs). It aims to expand available drug target space by providing improved methodology for the identification of inhibitors for such binding sites and which represent the majority of potential drug targets. The main goal of this paper is to provide a methodological overview of the use and application of the REPLACE strategy which involves computational and synthetic chemistry approaches. REPLACE is exemplified through its application to the development of non-ATP competitive cyclin dependent kinases (CDK) inhibitors as anti-tumor therapeutics. CDKs are frequently deregulated in cancer and hence are considered as important targets for drug development. Inhibition of CDK2/cyclin A in S phase has been reported to promote selective apoptosis of cancer cells in a p53 independent manner through the E2F1 pathway. Targeting the protein-protein interaction at the cyclin binding groove (CBG) is an approach which will allow the specific inhibition of cell cycle over transcriptional CDKs. The CBG is recognized by a consensus sequence derived from CDK substrates and tumor suppressor proteins termed the cyclin binding motif (CBM). The CBM has previously been optimized to an octapeptide from p21Waf (HAKRRIF) and then further truncated to a pentapeptide retaining sufficient activity (RRLIF). Peptides in general are not cell permeable, are metabolically unstable and therefore the REPLACE (REplacement with Partial Ligand Alternatives through Computational Enrichment) strategy has been applied in order to generate more drug-like inhibitors. The strategy begins with the design of Fragment ligated inhibitory peptides (FLIPs) that selectively inhibit cell cycle CDK/cyclin complexes. FLIPs were generated by iteratively replacing residues of HAKRRLIF/RRLIF with fragment like small molecules (capping groups), starting from the N-terminus (Ncaps), followed by replacement on the C-terminus. These compounds are starting points for the generation of non-ATP competitive CDK inhibitors as anti-tumor therapeutics.

Abstract 2431: Co-targeting ROS and CLU-mediated stress response using SMIP004 (a novel inducer of ROS and cancer cell selective apoptosis) and OGX-011 in MDV3100-resistant, castrate-resistant prostate cancer

Abstract Prostate cancer is the most common male carcinoma in North America and the second leading cause of cancer related death in men. While new drugs targeting Androgen Receptor (AR), like abiraterone and MDV3100, significantly increase survival of patients with castration resistant prostate cancer (CRPC), many patients still die of metastatic resistant disease. Continued therapeutic improvements towards prolonging the chronicity of CRPC will require characterization of innate and stress-activated survival responses, and rational combinatorial co-targeting strategies designed to abrogate them. OGX-011 is a second generation antisense oligonucleotide targeting Clusterin (CLU) currently under evaluation in phase III clinical trials. CLU is a molecular chaperone involved in endoplasmic reticulum (ER) stress, protein homeostasis and in many signaling and transcriptional survival networks. It is cytoprotective under stress conditions and correlated with treatment resistance in prostate and other cancers. SMIP004 is a novel drug that modulates cell cycle and ER stress and unfolded protein responses (UPR). Some of its fuctions have been correlated to Reactive Oxygen Species (ROS) modulation. However, the exact mechanisms of action of SMIP004 are not completely understood. Cell survival of LNCaP (castration sensitive), C-42 (castration resistant), M42D, M49F (MDV3100 resistant) and PC3 (AR negative) human prostate cancer cell lines was assessed by crystal violet assay. CLU silencing was obtained by transfecting cells with short interference RNA or OGX011. Western blotting analysis was used to detect proteins expression and RT-QPCR for mRNA levels. ROS were evaluated in live cells after incubation with 2′,7′-DCFDA by FACS Canto II. Consistent with induction of proteotoxic stress, SMIP004 significantly up-regulated CLU mRNA and protein levels in prostate cancer cell lines sensitive and resistant to MDV3100. SMIP004 as well as MDV3100 were able to increase ROS production. Interestingly CLU silencing, through siRNA or OGX011, inhibited ROS while CLU overexpression, after SMIP004 and/or MDV3100, significantly induced ROS production in MDV3100 sensitive and resistant cells. Moreover SMIP004 efficiently controlled cell survival of MDV3100 sensitive and resistant prostate cancer cells but the combination with clusterin inhibitors had a synergistic effect in these cells. Interestingly, SMIP004 reduced AR variant-7, a AR splicing variant associated with abiraterone and MDV3100 resistance, in both MDV3100 sensitive and resistant cells. Clusterin plays a central role in ROS tolerance in prostate cancer cells. Clusterin inhibition in combination with SMIP004 is able to control cell survival of MDV-3100 sensitive and resistant cells also trough a modulation of oxydative stress. Citation Format: Lucia Nappi, Eliana Beraldi, Fan Zhang, Dieter Wolf, Martin Gleave. Co-targeting ROS and CLU-mediated stress response using SMIP004 (a novel inducer of ROS and cancer cell selective apoptosis) and OGX-011 in MDV3100-resistant, castrate-resistant prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2431. doi:10.1158/1538-7445.AM2015-2431

Non genomic loss of function of tumor suppressors in CML: BCR-ABL promotes IκBα mediated p53 nuclear exclusion.

Tumor suppressor function can be modulated by subtle variation of expression levels, proper cellular compartmentalization and post-translational modifications, such as phosphorylation, acetylation and sumoylation. The non-genomic loss of function of tumor suppressors offers a challenging therapeutic opportunity. The reactivation of a tumor suppressor could indeed promote selective apoptosis of cancer cells without affecting normal cells. The identification of mechanisms that affect tumor suppressor functions is therefore essential. In this work, we show that BCR-ABL promotes the accumulation of the NFKBIA gene product, IκBα, in the cytosol through physical interaction and stabilization of the protein. Furthermore, BCR-ABL/IκBα complex acts as a scaffold protein favoring p53 nuclear exclusion. We therefore identify a novel BCR-ABL/IκBα/p53 network, whereby BCR-ABL functionally inactivates a key tumor suppressor.

Systemic Administration of Platelets Incorporating Inactivated Sendai Virus Eradicates Melanoma in Mice

Tumor microenvironments include a number of fibrin clots due to the microbleeding caused by cancer cell invasion into blood vessels, which suggests the potential utility of a platelet vector for systemic cancer treatment. We previously reported that inactivated Sendai virus (hemagglutinating virus of Japan; HVJ) envelope (HVJ-E) activates anti-tumor immunity and induces cancer cell-selective apoptosis. The hemagglutination activity that blocks the systemic administration of HVJ-E was dramatically attenuated by incorporation into platelets. Platelets incorporating HVJ-E (PH complex) were then injected into the tail veins of B16F10 melanoma-bearing mice. The PH complex primarily accumulated in tumor tissues and caused the significant accumulation of various immune cells in the tumor bed. Injections of the PH complex to the melanoma-bearing mouse significantly reduced the tumor size, and the tumor growth was ultimately arrested. Secretion of the chemokine regulated upon activation normal T-expressed and presumably secreted (RANTES) was upregulated following PH stimulation. The RANTES-depletion in melanoma-bearing mice significantly attenuated the cytotoxic T lymphocyte activity and led to a dramatic abrogation of the mouse melanoma suppression induced by the PH complex. Thus, a platelet vector incorporating viral particles, a Trojan horse for cancer treatment, will provide a new approach for cancer therapy using oncolytic viruses.

Melanoma differentiation associated gene-7/interleukin-24 induces caspase-3 denitrosylation to facilitate the activation of cancer cell apoptosis.

Melanoma differentiation-associated gene-7 (mda-7)/interleukin-24 (IL-24) induces caspase-3 cleavage and subsequent activation via the intrinsic or extrinsic pathway to result in cancer cell-selective apoptosis, but whether mda-7/IL-24 may directly regulate caspase-3 through the post-translational modification remains unknown. Here, we reported that tumor-selective replicating adenovirus ZD55-IL-24 led to caspase-3 denitrosylation and subsequent activation, indicating that caspase-3 denitrosylation played a crucial role in ZD55-IL-24-induced cancer cell apoptosis. To confirm the relationship between caspase-3 denitrosylation and its activation in response to ZD55-IL-24, we treated carcinoma cells with the different nitric oxide (NO) regulators to modulate caspase-3 denitrosylation level, then observed the corresponding caspase-3 cleavage. We found that NO inhibitor 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxy-3-oxide (PTIO) promoted caspase-3 denitrosylation and caspase-3 cleavage, thereby exacerbating ZD55-IL-24-induced cancer cell apoptosis, whereas NO donor sodium nitroprusside (SNP) showed the opposite effect. Moreover, caspase-3 denitrosylation facilitated its downstream target poly ADP-ribose polymerase (PARP) degradation that further increased the apoptotic susceptibility. Although caspase-3 activation controlled by denitrosylation modification has emerged as an important regulator of programmed cell death, the detailed molecular mechanism by which caspase-3 exerts its denitrosylation modification in response to ZD55-IL-24 still needs to be elucidated. Thus, our results demonstrated that ZD55-IL-24 increased Fas expression to enhance thioredoxin reductase 2 (TrxR2), which was responsible for caspase-3 denitrosylation. Collectively, these findings elucidate that ZD55-IL-24 induces caspase-3 denitrosylation through Fas-mediated TrxR2 enhancement, thereby facilitating caspase-3 cleavage and the downstream caspase signaling pathway activation, which provides a novel insight into ZD55-IL-24-induced cancer-specific apoptosis by post-translational modification of the apoptotic executor caspase-3.

Small Molecule-Induced Mitochondrial Disruption Directs Prostate Cancer Inhibition via Unfolded Protein Response Signaling

We previously identified SMIP004 (N-(4-butyl-2-methyl-phenyl) acetamide) as a novel inducer of cancer-cell selective apoptosis of human prostate cancer cells. SMIP004 decreased the levels of positive cell cycle regulators, upregulated cyclin-dependent kinase inhibitors, and resulted in G1 arrest, inhibition of colony formation in soft agar, and cell death. However, the mechanism of SMIP004-induced cancer cell selective apoptosis remained unknown. Here, we used chemical genomic and proteomic profiling to unravel a SMIP004-induced pro-apoptotic pathway, which initiates with disruption of mitochondrial respiration leading to oxidative stress. This, in turn, activates two pathways, one eliciting cell cycle arrest by rapidly targeting cyclin D1 for proteasomal degradation and driving the transcriptional downregulation of the androgen receptor, and a second pathway that activates pro-apoptotic signaling through MAPK activation downstream of the unfolded protein response (UPR). SMIP004 potently inhibits the growth of prostate and breast cancer xenografts in mice. Our data suggest that SMIP004, by inducing mitochondrial ROS formation, targets specific sensitivities of prostate cancer cells to redox and bioenergetic imbalances that can be exploited in cancer therapy.

Alpha-Tocopheryl Succinate Inhibits Autophagic Survival of Prostate Cancer Cells Induced by Vitamin K3 and Ascorbate to Trigger Cell Death

BackgroundThe redox-silent vitamin E analog α-tocopheryl succinate (α-TOS) was found to synergistically cooperate with vitamin K3 (VK3) plus ascorbic acid (AA) in the induction of cancer cell-selective apoptosis via a caspase-independent pathway. Here we investigated the molecular mechanism(s) underlying cell death induced in prostate cancer cells by α-TOS, VK3 and AA, and the potential use of targeted drug combination in the treatment of prostate cancer.Methodology/Principal FindingsThe generation of ROS, cellular response to oxidative stress, and autophagy were investigated in PC3 prostate cancer cells by using drugs at sub-toxic doses. We evaluated whether PARP1-mediated apoptosis-inducing factor (AIF) release plays a role in apoptosis induced by the combination of the agents. Next, the effect of the combination of α-TOS, VK3 and AA on tumor growth was examined in nude mice. VK3 plus AA induced early ROS formation associated with induction of autophagy in response to oxidative stress, which was reduced by α-TOS, preventing the formation of autophagosomes. α-TOS induced mitochondrial destabilization leading to the release of AIF. Translocation of AIF from mitochondria to the nucleus, a result of the combinatorial treatment, was mediated by PARP1 activation. The inhibition of AIF as well as of PARP1 efficiently attenuated apoptosis triggered by the drug combination. Using a mouse model of prostate cancer, the combination of α-TOS, VK3 and AA was more efficient in tumor suppression than when the drugs were given separately, without deleterious side effects.Conclusions/Significanceα-TOS, a mitochondria-targeting apoptotic agent, switches at sub-apoptotic doses from autophagy-dependent survival of cancer cells to their demise by promoting the induction of apoptosis. Given the grim prognosis for cancer patients, this finding is of potential clinical relevance.

Highlights of This Issue

Immunosuppression in glioblastoma is a critical barrier to effective immunotherapy. Wainwright and colleagues found that overexpression of indoleamine 2,3 dioxygenase (IDO) in glioma was associated with a decreased lifespan in patients. Using orthotopic and transgenic models of glioma, as well as shRNA-mediated knockdown, they then showed that glioma-derived IDO expression led to increased tumor-infiltrating regulatory T cells (Treg) and decreased overall survival. In contrast, IDO inhibition led to decreased Treg levels and increased longterm survival. This study provides a strong rationale for further analysis of IDO in the context of malignant glioma, with a new focus on therapeutic inhibition.The vast majority of human surgical samples are stored as formalin-fixed, paraffin-embedded (FFPE) blocks, with more than 20 million archived annually in the United States. Although this represents a tremendous resource for disease research, RNA degradation has limited their use for transcriptional profiling. Waldron and colleagues analyzed gene expression in more than 1,000 FFPE samples from long-term multicenter studies of breast and colorectal cancer, together with a meta-analysis including 12 additional datasets. They introduce quality control protocols that improve discovery of reproducible biomarkers, an essential step forward in realizing the potential of clinical FFPE tissues for genomic analysis.The miRNA regulatory network is emerging as a novel target for the treatment of human cancer. To explore the potential of miR-34a against human multiple myeloma, Di Martino and colleagues evaluated the therapeutic activity triggered by miR-34a mimics. The translational relevance of the study resides in the finding that systemic delivery of novel lipidic-formulated miR-34a exerts antimyeloma activity in different clinically relevant murine models of human multiple myeloma, without toxicity. The specificity of miR-34a activity was shown by downregulation of canonical targets BCL2 and CDK6. These findings provide a framework for clinical development of formulated miR-34a mimics in multiple myeloma.The treatment of cancer with oncolytic viruses primarily depends on the selective viral replication in cancer cells. However, a replication-incompetent Sendai virus (HVJ) envelope (HVJ-E) showed effective oncolytic activities without producing toxic effects in nonmalignant cells. Matsushima-Miyagi and colleagues elucidated the mechanism for cancer cell–selective apoptosis induced by HVJ-E. The viral RNA fragments derived from HVJ-E upregulated TRAIL and Noxa selectively in cancer cells, but not in noncancerous cells, through the RIG-I/MAVS signaling pathway. The orthotopic prostate cancers were dramatically eradicated in immunodeficient NOD–SCID mice injected with HVJ-E. Thus, the RIG-I/MAVS signaling pathway represents an attractive target for cancer therapy.

Evaluation of KPT-SINE (Selective Inhibitors of CRM1-Mediated Nuclear Export) in AML and T-ALL.

e13586 Background: CRM1 is the major nuclear exporter that mediates transport of a variety of molecules, including proteins involved in tumor suppressor and cellular proliferation pathways. The crystal structure of CRM1, in complex with its export cargo, Snurportin 1, has been recently resolved. The molecular understanding of the CRM1-cargo binding interface has led to the development of novel small molecule inhibitors of CRM1-cargo interaction, termed KPT-Selective Inhibitors of Nuclear Export (SINE). KPT-SINE are potent, drug-like CRM1 inhibitors that irreversibly inactivate the CRM1-directed protein export by covalent modification of the essential CRM1-cargo binding residue Cys528. The inhibition of the CRM1 nuclear export has been shown to lead to selective apoptosis in cancer cells when compared to normal cells. Here, we assess the efficacy of the KPT-SINE in human AML, T-ALL, and normal hematopoietic cells. Methods: The viability of a panel of human AML and T-ALL cell lines upon treatment by the KPT-SINE was assessed. Dose-response measurements were done using serial dilutions of KPT-SINE from 1 µM to 0.3 nM and luminescent cell viability assay. Apoptosis was measured using Annexin V staining and TUNEL assays. Results: KPT-SINE induces rapid apoptosis in 10 of 14 AML and 12 of 15 T-ALL cell lines with IC50s of 15-100 nM. In the KPT-SINE-sensitive cell lines, BCL2 overexpression suppresses KPT-SINE-induced apoptosis, indicating its intrinsic pathway mediation. Importantly, KPT-SINE compounds, either KPT-251 at 75 mg/kg or KPT-330 at 15 or 25 mg/kg, induced remarkable growth suppression in MV4-11 human AML cells engrafted into immunodeficient NSG mice with minimal toxicity to normal mouse hematopoietic cells after 35 days of treatment. Bone marrow biopsies of KPT-treated mice were remarkable in that they showed normal hematopoietic cell morphology and cellularity after 35 days of treatment. Xenograft studies to address the potency of the KPT-251 and KPT-330 on MOLT-4 human T-ALL cells in vivo are ongoing. Conclusions: These studies emphasize the clinical promise of the KPT-SINE as a novel and selective drug candidate for the treatment of AML and T-ALL.

Abstract 1815: CRM-1 as a potential therapeutic target in pancreatic cancer

Abstract CRM-1 exportin protein encoded by the XPO-1 gene maintains cancer cells through nuclear export signal (NES)-dependent protein exclusion of tumor suppressor proteins (TSPs). High CRM-1 expression has been correlated with poor prognosis in pancreatic cancer making it an attractive therapeutic target. Numerous attempts have been made in the past to develop specific CRM-1 inhibitors; however, earlier inhibitors such as Leptomycin B failed in the clinic due to acute off target toxicity. Using structure based drug design; we have identified new class of selective inhibitors of nuclear export (SINE) that irreversibly inhibits CRM1 and lock TSPs in cancer cell nucleus leading to cancer cell selective apoptosis. The inhibitors have excellent pharmacokinetic parameters and minimal toxicity to normal tissue. Here, we tested SINEs against pancreatic cancer, a disease that urgently needs novel and effective therapies. CRM-1 inhibitors [(KPT-185 (most potent analog), KPT-127, KPT-205, KPT-227 as well as Leptomycin B (as positive control) and not its inactive analog KPT-Tran (as negative control)] induced growth inhibition (MTT) and apoptosis (Annexin V and Histone DNA ELISA) in five cell lines (IC50 ∼150 nM). KPT-SINEs did not have any effect on normal HPDE cells (IC50>5 µM). Mechanistically, KPT-185 mediated apoptosis was found to be through activation, phosphorylation and nuclear localization of a TSP prostate apoptosis response 4 (PAR-4). PAR-4 has earlier been established by our laboratory as a therapeutic target in pancreatic cancer. Co-immunoprecipitation studies demonstrated that KPT-185 dissociated CRM-1-PAR-4 and CRM-1-p-PAR-4 interaction. Further, PAR-4 siRNA knockdown abrogated KPT-SINEs ability to induce growth inhibition or apoptosis. In normal cells PAR-4 localization or phosphorylation was not observed and this was associated with low basal expression of PAR-4 and its phosphorylation inducer protein kinase A protein. Role of other TSPs such as p53 and p27 was also explored, however, our investigations directly point to a PAR-4 specific mechanism. KPT-251 (structurally very similar to KPT-185 with improved pharmacokinetics properties) was well tolerated in SCID mice upto 150 mg/kg, p.o. Oral administration of analog KPT-251 at 25 and 150 mg/kg daily for two weeks reduced pancreatic tumors significantly (p<0.01). Histological examination of remnant tumors showed enhanced staining of PAR-4 and reduced Ki67. Tumor tissue lysates were probed for protein expression using western blotting that revealed activation of PAR-4 signaling and co-immunoprecipitation assay demonstrated dissociation of CRM-1-PAR-4 in tumors as well. In conclusion, discovery of our novel non-toxic KPTs can significantly impact the advancement of the field of CRM-1 inhibitors and could become effective anti-cancer therapeutics for pancreatic cancer as well as other solid tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1815. doi:1538-7445.AM2012-1815

Selective toxicity on canine blood cells by using atmospheric-pressure plasma jets

Atmospheric pressure plasma jets have received increasing attention due to their non-thermal effects on biological samples. Their noticeable sterilization on tissues, selective apoptosis of cancer cells and effective regeneration of damaged tissues have successfully demonstrated their medical applicability. However, the side effects or toxic effects on normal cells should be investigated fully before real applications. In this study, we report the selective toxicity of atmospheric pressure plasma jets (APPJ) on blood cells in vitro for the first time. A complete blood cell count is used to count and size the blood cells statistically. We found gas- and time-dependent effects of APPJ exposure on the number ratios and the size distributions of red blood cells, white blood cells and platelets.

KPT-SINE, a Potent, Small Molecule Inhibitor of CRM1-Dependent Nuclear-Cytoplasmic Shuttling, with Potent Activity Against T-ALL and AML

Abstract 2622 Background:CRM1 (XPO1) is the major nuclear exporter that mediates transport of a variety of cargos, including proteins involved in tumor suppressor and cellular proliferation pathways (e.g. p53, PI3K/AKT, and NF-kB). CRM1 is upregulated in a range of solid and hematologic malignancies and its overexpression is correlated with poor prognosis, and with resistance to chemotherapy. Recently, the crystal structure of CRM1, in complex with its export cargo, Snurportin 1, has been resolved. The structure elucidates the key contact residues required for the formation of the CRM1-cargo export complex. The molecular understanding of the CRM1-cargo binding interface has led to the development of novel small molecule inhibitors of CRM1-cargo interaction, termed KPT-Selective Inhibitors of Nuclear Export (SINE). KPT-SINE are potent, drug-like CRM1 inhibitors that irreversibly inactivate the CRM1-directed protein export by covalent modification of the essential CRM1-cargo binding residue Cys528. The inhibition of the CRM1 nuclear export has been shown to lead to selective apoptosis in cancer cells when compared to normal cells. Here, we assess the efficacy of the KPT-SINE in human T-ALL, AML, and normal hematopoietic cells. Methods: The viability of a panel of human T-ALL and AML cell lines and normal CD34+ progenitor cells upon treatment by the KPT-SINE was assessed. Dose-response measurements were done using serial dilutions of KPT-SINE from 1 μM to 0.3 nM and luminescent cell viability assay. Apoptosis was measured using Annexin V staining and TUNEL assays. Results: KPT-SINE induces rapid apoptosis in 12 of 15 T-ALL and 10 of 14 AML cell lines with 50% inhibitory concentrations (IC50s) of 15–100 nM. In the KPT-SINE-sensitive cell lines, BCL2 overexpression suppresses KPT-SINE-induced apoptosis, indicating its intrinsic pathway mediation. Importantly, KPT-SINE exhibits selective cytotoxicity against tumor cells when compared to peripheral blood mononuclear cells (PBMCs) and CD34+ progenitor cells in vitro. KPT-SINE at 57 and 150 mg/kg induced 100% inhibition of tumor growth in vivo in a MOLT-4 T-ALL xenograft study. Other xenograft studies to address the potency of the KPT-SINE on selected AML lines in vivo are ongoing. These studies emphasize the clinical promise of the KPT-SINE as a novel and selective drug candidate for the treatment of T-ALL and AML. Disclosures:McCauley:Karyopharm Therapeutics: Employment. Kauffman:Karyopharm: Equity Ownership. Shacham:Karyopharm: Equity Ownership.

Abstract 1351: Computational design and synthesis of non-peptidic inhibitors targeting the cyclin binding groove of CDK2/Cyclin A

Abstract Inhibition of CDK2/Cyclin A (CDK2/CA) at G1/S phase of cell cycle controls cell proliferation and induces selective apoptosis of cancer cells. Neutralization of E2F-1by CDK2/CA phosphorylation is required prior to S-phase exit or apoptosis is triggered. E2F-1 is deregulated in many types of cancer and inhibition of CDK2/CA leads to selective cell death in these tumors. In our present study we target the substrate recruitment site knows as cyclin binding groove (CBG) for inhibition of CDK2/CA. The CBG recognizes a consensus sequence present on cyclin interacting proteins and the endogenous tumor suppressors knows cyclin binding motif (CBM) which has been minimized to the pentapeptide, RRLIF. It has been shown that the drug likeliness of the peptide can be improved by ligation of small molecules to truncated sequences. This was achieved using a unique strategy for designing inhibitors of protein-protein interactions known as REPLACE, in which small molecule fragments were identified by in silico methods. These were then ligated onto a truncated peptide sequence in order to replace critical residues, including the N-terminal Arginine moiety important for cyclin binding. Small molecule fragments for in silico docking were selected based on molecule weight, drug like features and possession of a carboxylic acid group for the formation of the peptide link. Docking was carried out using LigandFit and the crystal structure of 1-phenyl-1H-[1,2,4]triazole N-terminally capped peptides (Ncaps) in the CBG provided insight into molecular design. The 3,5 dichloro1-phenyl-1H-[1,2,4]triazole derivative linked to Arg-Leu-Asn-pF-Phe was previously shown to be a potent inhibitor with micromolar activity. In our present study we have replaced the N-terminal Arginine using different heterocyclic isosteres capable of interactions similar to critical amino acids of the parent peptide and the triazole. In order to probe the structure-activity relationship, we synthesized pyrazole, furan, pyrrole and thiazole systems and explored various substitutions of the phenyl ring. A number of Ncaps were then ligated to the tetra peptide, RLIF using solid phase synthesis, purified by reverse phase HPLC and characterized by MS. In vitro binding and functional assays were performed in order to study the inhibitory effect of compounds on CDK2/Cyclin A prior to further evaluation in cell viability assays to determine anti-tumor effects. On the basis of the results, further high throughput docking of potential heterocyclic fragments was carried out to identify N-capping groups of varying chemical diversity for synthesis and in vitro testing. The results presented demonstrate the utility of the REPLACE method in the development of protein-protein interaction inhibitors and non-ATP competitive CDK2 inhibitors as anti-tumor therapeutics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1351. doi:10.1158/1538-7445.AM2011-1351

Abstract SY08-02: Markers of caspase activation in cells and serum

Abstract The final and hoped for outcome for treatment with cancer drugs is selective apoptosis of cancer cells. However, despite the low response rates to cancer drugs patients do not discover this until months after treatment has begun. Apoptosis is rapid process which is often induced within 24 hours of drug treatment. The hallmark of apoptosis is the activation of caspases, cysteine proteases that are specific for cleaving target proteins after aspartic acid. We have developed a novel proteomics technology that allows us to tag and enrich for caspase-cleaved proteins during apoptosis (Mahrus et al (2008) Cell, 134, 866-876. “Global Sequencing of Proteolytic Cleavage Sites in Apoptosis by Specific Labeling of Protein N Termini”). We have been characterizing the apoptotic products of caspase cleavage in a variety of cell lines and in human serum with the view that this information can be used to monitor apoptosis during cancer drug treatment. These and other studies in a variety of cancer cell lines showed that upwards of 1000 proteins are cleaved by caspases during apoptosis. However, in any given cell line we find there are fewer than 500 targets because of differences in expression patterns between cell types. Thus, while many targets are in common from one cell line to another, many are unique. We have developed single-reaction monitoring (SRM) methods to identify these products in serum. We are beginning to identify specific apoptotic products in leukemia patients treated with various cancer drugs. These studies and the prospects for using these as apoptotic markers to assist in cancer drug treatment will be discussed. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr SY08-02. doi:10.1158/1538-7445.AM2011-SY08-02

Is There a Role for Nuclear Factor κB in Tumor Necrosis Factor‐Related Apoptosis‐Inducing Ligand Resistance?

One strategy for cancer management consists of promoting selective apoptosis of cancer cells. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a proapoptotic cytokine, is a promising anticancer agent because of its ability to selectively induce apoptosis in established tumor cell lines but not in nontransformed cells. However, many tumors have developed mechanisms of resistance against killing by TRAIL. Whether or not the transcription factor nuclear factor (kappaB) is involved in TRAIL resistance is uncertain, and this short review aims to summarize currently available data on this question.

Malonyl-CoA decarboxylase inhibition is selectively cytotoxic to human breast cancer cells

Fatty acid synthase (FAS) inhibition initiates selective apoptosis of cancer cells both in vivo and in vitro, which may involve malonyl-CoA metabolism. These findings have led to the exploration of malonyl-CoA decarboxylase (MCD) as a potential novel target for cancer treatment. MCD regulates the levels of cellular malonyl-CoA through the decarboxylation of malonyl-CoA to acetyl-CoA. Malonyl-CoA is both a substrate for FAS and an inhibitor of fatty acid oxidation acting as a metabolic switch between anabolic fatty acid synthesis and catabolic fatty acid oxidation. We now report that the treatment of human breast cancer (MCF7) cells with MCD small interference RNA (siRNA) reduces MCD expression and activity, reduces adenosine triphosphate levels, and is cytotoxic to MCF7 cells, but not to human fibroblasts. In addition, we synthesized a small-molecule inhibitor of MCD, 5-{(Morpholine-4-carbonyl)-[4-(2,2,2-trifluoro-1-hydroxy-1-trifluoromethyl-ethyl)-phenyl]-amino}-pentanoic acid methyl ester (MPA). Similar to MCD siRNA, MPA inhibits MCD activity in MCF7 cells, increases cellular malonyl-CoA levels and is cytotoxic to a number of human breast cancer cell lines in vitro. Taken together, these data indicate that MCD-induced cytotoxicity is likely mediated through malonyl-CoA metabolism. These findings support the hypothesis that MCD is a potential therapeutic target for cancer therapy.

Using Noval Experimental PM 701 in Refractory Hodgkin's Disease Combined with conventional Therapy

Background: To the many profound changes we now face in health care, Complementary and Alternative Medicine (CAM) must be added as a major factor. There is evidence of the increasing use of CAM by patients diagnosed with cancer. Interest in CAM continues to grow at an exponential rate despite the advances made by conventional medicine. Given the increasing desire of cancer patients to use CAM, it is important that clinicians have a good understanding of the evidence available in this field and build a trust with their patients. Many oncologists are not familiar with CAM used by many of their patients.Aims: To report the effectiveness and safety of a natural experimental agent used with conventional therapy in a patient with Stage IV Hodgkin's lymphoma refractory to treatment.Methods: An eight-years-old Saudi girl was diagnosed as Stage IV Hodgkin's lymphoma with lung infiltration. The patient was started on chemotherapy (ABVD), and needed prolonged hospitalization due to weakness, loss of appetite, cachexia, dyspnea, cough, fever, and marked hepatosplenomegaly. She had six cycles of chemotherapy with a slight improvement in her general condition. However, she as considered refractory to conventional therapy. We were planning to refer her for bone marrow transplant. The patient was sent home to be readmitted for the seventh cycle of ABVD. Unfortunately, the patient's general condition deteriorated and she was admitted to the Intensive Care Unit (ICU), with acute Respiratory Distress Syndrome (RDS). She was markedly unstable and required respiratory ventilation support. After discussions involving the parents, it was suggested to use an experimental natural remedy of proven efficacy in inducing apoptosis in tissue cultures and animals in our laboratories. On the fifth day in ICU, the patient was initiated with full dose ABVD combined with the natural remedy, classified as PM 701. Within 2 days, the patient became fully conscious, showed improved general condition and returned to the pediatric ward to continue her treatment.Results: After the seventh cycle of chemotherapy and the use of natural remedy, her general condition improved markedly with significant reduction of the hepatosplenomegaly and pleural effusions. Repeated CT scans of chest and abdomen showed marked regression of her disease. She had one more cycle of chemotherapy combined with PM 701 and assessment after the eighth cycle showed complete remission of her disease including in the lungs. This natural agent has been fully studied with animal models at the King Fahed Medical Research Center's laboratory and showed potent selective apoptosis of cancer cells, effective and safe in animal models.Summary: Most childhood malignancy is curable with combined intensive conventional chemotherapy, however up to 30% may have relapse or be resistant to chemotherapy. We report in this case successful management with the combination of conventional therapy with experimental medicine (PM 701) in the treatment of refractory Hodgkin's lymphoma, which needs further control studies in the future to prove the efficacy and safety to be used on oncology patients. Integration between chemotherapy and natural CAM should be under close supervision to ensure safety and effectiveness. Scientific research needs more effort to support this line of medicine.

A new era of cancer therapy: Cancer cell targeted therapies are coming of age

The large majority of today's cancer therapies are based on the removal of solid tumor masses (for example by surgery when possible) and a plethora of chemical or physical treatments such as chemo- and radiotherapy which induce death of particularly sensitive or rapidly growing cells. These approaches are variously combined in order to optimize therapeutic indices. While in some cases, such as childhood leukemia, these treatments may cure patients, it is common knowledge that they are associated with serious side effects. The main conceptual reason for this is that neither cancer cells nor the cancer cause(s) are directly targeted. In addition to a high toxicity and a low quality of life, these traditional therapies can give rise to therapy-induced secondary cancers. Here we will neither discuss the various optimization possibilities nor arguments for and against established therapies. Rather we want to reflect about recent advances, challenges and perspectives of cancer therapeutic concepts which aim at increasing cancer-selectivity. More precisely, we will discuss two such concepts from a cell biological and molecular oncology perspective, namely to (i) target the cause of the cancer and (ii) to (re)activate specific endogenous pathways for cancer cell-selective apoptosis.