New Class Of Anticancer Therapeutics Research Articles

Cationic Lipid Modification of Nuclear Hormone Receptor-Ligands: A Strategy for Developing a New Class of Targeted Anticancer Therapeutics

Ubiquitous expression patterns of nuclear hormone and their receptors (NHRs) have their own roles in performing essential functions in controlling hormone responsive element-promoted gene transcription. In diseased states such as cancer, NHRs play crucial roles and hence targeting these receptors are essential as it gives us a window of opportunity for developing targeted anticancer therapeutics. In comparison to traditional chemotherapy drugs, targeted therapeutic drugs are hypothetically advantageous in terms of efficacy and safety and hence the idea of developing such targeted drugs are inclined to become a mainstream cancer treatment option. This review selectively compiles data regarding NHR-targeting, while majorly focusing on cancer treatment using anticancer small molecules and/or nanotherapeutics targeting estrogen receptor, progesterone receptor, glucocorticoid receptor, and vitamin D receptor. In this study, we selectively emphasized on estrogen receptor. Herein, we mainly highlight the strategy of lipid-modification to convert respective receptor ligands into NHR-targeted anticancer molecules as well as nanotherapeutics. We focused on the strategy of chemical conjugation of those ligands with twin-aliphatic carbon chain-based cationic lipids. The strategy successfully led to the development of a new class of anticancer therapeutics. These are either small-molecule anticancer agents or selfaggregating nanotherapeutics. In spite of great anticancer output, the concept of NHR-targeted anti-cancer therapy still needs to overcome further hurdles before those are projected for clinical settings.

Abstract 1919: Activity and affinity tuning next-generation immunotoxins for targeted therapy

Abstract Classic cancer-associated receptors, such as EGFR and Her2, are often overexpressed on malignant cells relative to healthy cells. Numerous molecules have been developed to leverage this differential expression with the goal of developing specific and safe targeted therapies, including antibody-drug-conjugates and immunotoxins. The general schematic of these molecules is to fuse toxic payloads -either small molecules or proteins - with a targeting moiety that binds to the cancer-specific receptor. Despite the general success of this approach, even low levels of these receptors on healthy tissues result in on-target/off-tumor toxicity thus limiting the dose of therapeutic molecules available to tumors. Targeted therapies with greater on-tumor efficacy and reduced toxicity are urgently needed in order to further advance therapeutic outcomes. To achieve this, we set out to exploit the unique features of bacterial toxins as highly modular protein delivery systems to design next-generation targeted therapies. We demonstrate the capacity of the immunotoxin platform to deliver various therapeutic proteins, with distinct activities from inhibiting protein synthesis to degrading oncogenic signaling pathways, such as the RAS/MAPK pathway. Further, we show how modifying the affinity, valency, and specificity of the receptor-binding moieties, as well as tuning the enzymatic activity of the toxic enzyme payloads interact to affect the on-target efficacy and off-target toxicity of the targeted molecules. Altogether, these data demonstrate the potential of immunotoxins as highly flexible and tunable platforms for the development of a new class of anti-cancer therapeutics. Citation Format: Huazhu (Peter) Liang, Greg L. Beilhartz, Shi Bo Cao, Roman A. Melnyk. Activity and affinity tuning next-generation immunotoxins for targeted therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1919.

Abstract 1349: Application of the DARPin® technology for specific targeting of tumor-associated MHC class I:peptide complexes

Abstract Development of biologics or cell therapeutics which target MHC class I:peptide (pMHC) complexes for recognition and elimination of tumor cells is hindered by low affinity, cross-reactivity or challenging biochemical properties of antibody- and T-cell receptor-based binders. We hypothesized that DARPin® proteins may be particularly effective in solving this problem due to structural characteristics of their antigen binding surface and excellent biophysical properties. Panels of binders highly specific for a given pMHC complex were isolated from DARPin® libraries through several rounds of selection and counter selection on the relevant or irrelevant but structurally similar pMHC complexes using ribosome display. DARPin® binders were successfully isolated against pMHC complexes composed of different MHC class I alleles with various peptides derived from either tumor associated antigens or non-self viral proteins. A selected panel of DARPin® binders specific to HLA-A2 molecule in association with SLLMWITQC (SLL peptide), a peptide derived from NY-ESO-1, was used to create bi-specific T-cell engagers containing another moiety binding to the epsilon component of the CD3 complex, thus allowing highly sensitive analysis of pMHC specificity and potential cross-reactivity. Using a number of cellular assays, including peptide pulsing of TAP-deficient T2 cells, we confirmed high specificity of selected DARPin® proteins to the HLA-A2:SLL complex. Architectural fine tuning and sequence engineering allowed us to further increase the potency of the selected candidates without compromising the specificity. This was manifested as effective T cell mediated activation by relevant DARPin® constructs in the presence of HLA-A2+/NY-ESO-1+ tumor cells. Furthermore, HLA-A2+/NY-ESO-1+ cells but not HLA-A2+/NY-ESO-1- cells were effectively killed in the presence of engineered HLA-A2:SLL-specific DARPin® T-cell engagers. The versatility of the DARPin® platform and the engineering experience gained with NY-ESO-1 did allow us to identify highly potent and specific DARPins® proteins for additional clinically attractive pMHC targets.Alanine and X-scan mutagenesis demonstrated that, in many cases, interactions with several peptide residues located across the entire peptide sequence are critical for DARPin® protein binding to the pMHC complex. These data suggest that peptide residues exposed outside of the MHC peptide binding grove create the focal point of MHC:peptide:DARPin® proteins interactions. Further molecular and cellular analysis of DARPin® protein specificity will be evaluated in order to de-risk for potential clinically relevant toxicity.In conclusion, we show that the DARPin® technology platform may be highly instrumental in developing a new class of anti-cancer therapeutics based on specific targeting of pMHC complexes presented selectively by cancer cells. Citation Format: Natalia Venetz, Sandra Müller, Tim Schulte, Stefanie Fischer, Maria Paladino, Nicole Pina, Nadir Kadri, Sandra Bruckmaier, Andreas Cornelius, Tanja Hospodarsch, Tatyana Sandalova, Victor Levitsky, Adnane Achour, Marcel Walser. Application of the DARPin® technology for specific targeting of tumor-associated MHC class I:peptide complexes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1349.

Discovery and Mechanistic Characterization of a Select Modulator of AhR-regulated Transcription (SMAhRT) with Anti-cancer Effects.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor and a member of the bHLH/PAS (basic Helix-Loop-Helix/Per-Arnt-Sim) family of proteins. The AhR was cloned and characterized for its role in mediating the toxicity of dioxins. Subsequent research has identified the role of AhR in suppression of cancer cell growth. We hypothesized that the AhR is a molecular target for therapeutic intervention in cancer, and that activation of the AhR by unique AhR ligands in cancer cells could have anti-cancer effects including induction of cell death. This study describes the discovery and characterization of a new class of anti-cancer agents targeting the AhR, that we designate as Select Modulators of AhR-regulated Transcription (SMAhRTs). We employed two independent small molecule screening approaches to identify potential SMAhRTs. We report the identification of CGS-15943 that activates AhR signaling and induces apoptosis in an AhR-dependent manner in liver and breast cancer cells. Investigation of the downstream signaling pathway of this newly identified SMAhRT revealed upregulation of Fas-ligand(FasL), which is required for AhR-mediated apoptosis. Our results provide a basis for further development of a new class of anti-cancer therapeutics targeting an underappreciated molecular target, the AhR.

Abstract 690: Application of the DARPin® technology for specific targeting of tumor-associated MHC class I:peptide complexes

Abstract Development of biologics or cell therapeutics which target MHC class I:peptide (pMHC) complexes for recognition and elimination of tumor cells is hindered by low affinity, cross-reactivity or challenging biochemical properties of antibody- and T-cell receptor-based binders. We hypothesized that DARPin® proteins may be particularly effective in solving this problem due to structural characteristics of their antigen binding surface and excellent biophysical properties. Panels of binders highly specific for a given pMHC complex were isolated from DARPin® libraries through several rounds of selection and counter selection on the relevant or irrelevant but structurally similar pMHC complexes using ribosome display. DARPin® binders were successfully isolated against pMHC complexes composed of different MHC class I alleles with various peptides derived from either tumor associated antigens or non-self viral proteins. A selected panel of DARPin® binders specific to HLA-A2 molecule in association with SLLMWITQC (SLL peptide), a peptide derived from NY-ESO-1, was used to create bi-specific T-cell engagers containing another moiety binding to the epsilon component of the CD3 complex, thus allowing highly sensitive analysis of pMHC specificity and potential cross-reactivity. Using a number of cellular assays, including peptide pulsing of TAP-deficient T2 cells, we confirmed high specificity of selected DARPin® proteins to the HLA-A2:SLL complex. This was manifested as effective activation of T-cells in the presence of relevant DARPin® constructs tested at a range of concentrations and HLA A2 positive cells pulsed with the SLL or irrelevant HLA A2 binding peptides. Furthermore, HLA-A2+/NY-ESO-1+ cells but not HLA-A2+/NY-ESO-1- cells were effectively killed in the presence of HLA-A2:SLL-specific T-cell engagers. Alanine scanning mutagenesis demonstrated that, in many cases, interactions with several peptide residues located across the entire peptide sequence are critical for DARPin® protein binding to the pMHC complex. These data suggest that peptide residues exposed outside of the MHC peptide binding grove create the focal point of MHC:peptide:DARPin interactions. Further molecular and cellular analysis of DARPin® protein specificity using additional molecular and cellular approaches will be presented in order to de-risk for potential clinically relevant toxicity which may result from off-target interactions with other pMHC complexes structurally resembling the tumor-associated pMHC complex. In conclusion, we show that the DARPin® technology platform may be highly instrumental in developing a new class of anti-cancer therapeutics based on specific targeting of pMHC complexes presented selectively by cancer cells. Citation Format: Marcel Walcer, Natalia Venetz, Tim Schulte, Stefanie Fischer, Nicole Bassler, Maria Paladino, Nicole Pina, Denis Villemagne, Sandra Bruckmaier, Andreas Cornelius, Tanja Hospodarsch, Loic Duffet, Tatjana Sandalova, Adnane Achour, Victor Levitsky. Application of the DARPin® technology for specific targeting of tumor-associated MHC class I:peptide complexes [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 690.

Discovery of a First-in-Class Potent Small Molecule Antagonist against the Adrenomedullin-2 Receptor.

The hormone adrenomedullin has both physiological and pathological roles in biology. As a potent vasodilator, adrenomedullin is critically important in the regulation of blood pressure, but it also has several roles in disease, of which its actions in cancer are becoming recognized to have clinical importance. Reduced circulating adrenomedullin causes increased blood pressure but also reduces tumor progression, so drugs blocking all effects of adrenomedullin would be unacceptable clinically. However, there are two distinct receptors for adrenomedullin, each comprising the same G protein-coupled receptor (GPCR), the calcitonin receptor-like receptor (CLR), together with a different accessory protein known as a receptor activity-modifying protein (RAMP). The CLR with RAMP2 forms an adrenomedullin-1 receptor, and the CLR with RAMP3 forms an adrenomedullin-2 receptor. Recent research suggests that a selective blockade of adrenomedullin-2 receptors would be therapeutically valuable. Here we describe the design, synthesis, and characterization of potent small-molecule adrenomedullin-2 receptor antagonists with 1000-fold selectivity over the adrenomedullin-1 receptor, although retaining activity against the CGRP receptor. These molecules have clear effects on markers of pancreatic cancer progression in vitro, drug-like pharmacokinetic properties, and inhibit xenograft tumor growth and extend life in a mouse model of pancreatic cancer. Taken together, our data support the promise of a new class of anticancer therapeutics as well as improved understanding of the pharmacology of the adrenomedullin receptors and other GPCR/RAMP heteromers.

Cytotoxic Conjugates of Fibroblast Growth Factor 2 (FGF2) with Monomethyl Auristatin E for Effective Killing of Cells Expressing FGF Receptors.

Antibody–drug conjugates (ADCs) are a new class of anticancer therapeutics that combine the selectivity of targeted treatment, ensured by monoclonal antibodies, with the potency of the cytotoxic agent. Here, we applied an analogous approach, but instead of an antibody, we used fibroblast growth factor 2 (FGF2). FGF2 is a natural ligand of fibroblast growth factor receptor 1 (FGFR1), a cell-surface receptor reported to be overexpressed in several types of tumors. We developed and characterized FGF2 conjugates containing a defined number of molecules of highly cytotoxic drug monomethyl auristatin E (MMAE). These conjugates effectively targeted FGFR1-expressing cells, were internalized upon FGFR1-mediated endocytosis, and, in consequence, revealed high cytotoxicity, which was clearly related to the FGFR1 expression level. Among the conjugates tested, the most potent was that bearing three MMAE molecules, showing that the cytotoxicity of protein–drug conjugates in vitro is directly dependent on drug loading.

Abstract 2938: Tumour-associated macrophage polarisation and re-education with oncolytic viruses

Abstract Tumor-associated macrophages (TAMs) promote key processes in tumour progression through their plasticity and capacity to become polarised by tumours to have an immunosuppressive phenotype. However, the plasticity of these cells provides an opportunity for therapeutic ‘reprogramming’ towards a classically activated inflammatory phenotype thus preventing tumour-associated immunosuppression and inducing anti-tumour immunity. Oncolytic viruses (OV) represent a new class of anti-cancer therapeutics that promote anti-tumour responses through selective tumour cell killing and the induction of systemic anti-tumour immunity. We propose that TAMs can be targeted using an OV, leading to altered macrophage polarization and a reduction in tumour growth and metastasis. Experimental Design: In-vitro, cytotoxicity, viral replication and cytokine expression was assessed in a panel of primary human and murine macrophages following exposure to Sephrevir, an ICP34.5-deleted oncolytic herpes-simplex virus currently in Phase 1/2a trials. In-vivo, murine TS1-PyMT cells were implanted into the mammary fat pads of female FVB mice to establish a syngeneic immunocompetent mouse model of breast cancer. Tumour growth and overall survival were assessed following administration of Sephrevir given either by intratumoural (I.T) or intravenous injection (I.V). Immune cell enumeration and protective immunity were assessed post-mortem by immunofluorescence and flow cytometry. Results: Both primary human and mouse macrophages served as a host for oncolytic viral replication. Sephrevir was also found to attenuate TAM phenotypes as evidenced by increased “M1” receptor signatures (e.g. CD40, CD80, CD86, MHC II), increased pro-inflammatory gene expression (e.g. IL-1, IL-6, IL-8, CXCL10, TNF-a) and increased nitric oxide production. I.T and I.V administration of Sephrevir led to marked tumour shrinkage as well as the recruitment of cytotoxic T cells. Moreover, TAM polarisation was evident based on a change in signature with TAMs becoming skewed to IL-12hi/IL-10lo and a change in the iNOS/arginase1 ratio. Conclusions: Our results demonstrate that Sephrevir is associated with in vivo anti-tumour effects and importantly can be used for manipulating the phenotype of the abundant macrophage population located within the tumour microenvironment. Citation Format: Munitta Muthana, Emer Murphy, Amy Kwan, Natalie Winder, Joe Conner. Tumour-associated macrophage polarisation and re-education with oncolytic viruses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2938. doi:10.1158/1538-7445.AM2017-2938

Simultaneously double infection of oncolytic viruses leads to addition of cell death induction in glioblastoma cell lines

Meeting abstract Malignant glioblastomas are highly aggressive tumors virtually resistant to available treatments and predominantly leading to death. The standard treatment is surgical resection followed by chemo-irradiation therapy. However, the tumor always gains resistance after initial response. An improvement of outcome seems only be possible by evaluating new and innovative therapeutic strategies. An approach that has gained much interest for cancer treatment in the last years is virotherapy with oncolytic viruses. They represent a new class of anticancer therapeutics that have the ability to kill different forms of tumor cells. For three of such viruses (Reovirus, RV; Parvovirus-H, PV and Newcastle-DiseaseVirus, NDV) a strong tumorlytic effect on glioma cells is described in vitro and in animal models [1]. It is known that the viruses can induce different kinds of cell death, like apoptosis or necroptosis, via different pathways [2-4]. To investigate the oncolytic capacity and the induction of cell death of RV, PV and NDV the glioblastoma cell line U87 was simultaneously infected with two viruses followed by analysis of cell survival by MTT-assay and the type of cell death by FACS staining for Annexin V and Propidium iodide. Moreover the cells were stained for virus proteins to search for double infection. We could show that in simultaneously double infected cells, while the oncolytic effect of the stronger virus will be dominant, both viruses do induce the virus specific cell death. These effects are completely complementing each other. Furthermore we could show that the viruses can replicate at the same time in the cells. Therefore we conclude that the use of two or more oncolytic viruses in glioblastoma therapy is a promising tool to add to the traditional therapy.

Anticancer efficacy of a nitric oxide-modified derivative of bifendate against multidrug-resistant cancer cells.

The development of multidrug resistance (MDR) not only actively transports a wide range of cytotoxic drugs across drug transporters but is also a complex interaction between a number of important cellular signalling pathways. Nitric oxide donors appear to be a new class of anticancer therapeutics for satisfying all the above conditions. Previously, we reported furoxan‐based nitric oxide‐releasing compounds that exhibited selective antitumour activity in vitro and in vivo. Herein, we demonstrate that bifendate (DDB)‐nitric oxide, a synthetic furoxan‐based nitric oxide‐releasing derivative of bifendate, effectively inhibits the both sensitive and MDR tumour cell viability at a comparatively low concentration. Interestingly, the potency of DDB‐nitric oxide is the independent of inhibition of the functions and expressions of three major ABC transporters. The mechanism of DDB‐nitric oxide appears to be in two modes of actions by inducing mitochondrial tyrosine nitration and apoptosis, as well as by down‐regulating HIF‐1α expression and protein kinase B (AKT), extracellular signal‐regulated kinases (ERK), nuclear factor κB (NF‐κB) activation in MDR cells. Moreover, the addition of a typical nitric oxide scavenger significantly attenuated all the effects of DDB‐nitric oxide, indicating that the cytotoxicity of DDB‐nitric oxide is as a result of higher levels of nitric oxide release in MDR cancer cells. Given that acquired MDR to nitric oxide donors is reportedly difficult to achieve and genetically unstable, compound like DDB‐nitric oxide may be a new type of therapeutic agent for the treatment of MDR tumours.

Myricetin arrests human telomeric G-quadruplex structure: a new mechanistic approach as an anticancer agent.

The use of small molecules to arrest G-quadruplex structure has become a potential strategy for the development and design of a new class of anticancer therapeutics. We have studied the interaction of myricetin, a plant flavonoid and a putative anticancer agent, with human telomeric G-quadruplex TTAGGG(TTAGGG)3 DNA. Reverse transcription PCR data revealed significant repression in hTERT expression in MCF-7 breast cancer cells upon increasing the concentration of myricetin. Further, we conducted a telomeric repeat amplification protocol assay to confirm the inhibition of telomerase by myricetin. Optical spectroscopic techniques like circular dichroism, UV spectroscopy and fluorescence spectroscopy revealed the formation of a stable myricetin-G-quadruplex complex. The thermodynamic parameters of myricetin-G-quadruplex complex formation, presented through isothermal titration calorimetry studies, indicate the binding process to be thermodynamically favorable. In addition, high resolution NMR spectroscopy in conjunction with molecular dynamics simulation is employed to provide detailed mechanistic insights into the binding in the myricetin-G-quadruplex complex at the atomic level. Our results thus propose a new mode of action of myricetin as an anticancer agent via arresting telomeric G-quadruplex structure.

Simultaneously double infection of oncolytic viruses leads to addition of cell death induction in glioblastoma cell lines

Meeting abstract Malignant glioblastomas are highly aggressive tumors virtually resistant to available treatments and predominantly leading to death. The standard treatment is surgical resection followed by chemo-irradiation therapy. However, the tumor always gains resistance after initial response. An improvement of outcome seems only be possible by evaluating new and innovative therapeutic strategies. An approach that has gained much interest for cancer treatment in the last years is virotherapy with oncolytic viruses. They represent a new class of anticancer therapeutics that have the ability to kill different forms of tumor cells. For three of such viruses (Reovirus, RV; Parvovirus-H, PV and Newcastle-DiseaseVirus, NDV) a strong tumorlytic effect on glioma cells is described in vitro and in animal models [1]. It is known that the viruses can induce different kinds of cell death, like apoptosis or necroptosis, via different pathways [2-4]. To investigate the oncolytic capacity and the induction of cell death of RV, PV and NDV the glioblastoma cell line U87 was simultaneously infected with two viruses followed by analysis of cell survival by MTT-assay and the type of cell death by FACS staining for Annexin V and Propidium iodide. Moreover the cells were stained for virus proteins to search for double infection. We could show that in simultaneously double infected cells, while the oncolytic effect of the stronger virus will be dominant, both viruses do induce the virus specific cell death. These effects are completely complementing each other. Furthermore we could show that the viruses can replicate at the same time in the cells. Therefore we conclude that the use of two or more oncolytic viruses in glioblastoma therapy is a promising tool to add to the traditional therapy.

Tumor Cell Death Mediated by Peptides That Recognize Branched Intermediates of DNA Replication and Repair

Effective treatments for cancer are still needed, both for cancers that do not respond well to current therapeutics and for cancers that become resistant to available treatments. Herein we investigated the effect of a structure-selective d-amino acid peptide wrwycr that binds replication fork mimics and Holliday Junction (HJs) intermediates of homologous recombination (HR) in vitro, and inhibits their resolution by HJ-processing enzymes. We predicted that treating cells with HJ-binding compounds would lead to accumulation of DNA damage. As cells repair endogenous or exogenous DNA damage, collapsed replication forks and HJ intermediates will accumulate and serve as targets for the HJ-binding peptides. Inhibiting junction resolution will lead to further accumulation of DNA breaks, eventually resulting in amplification of the damage and causing cell death. Both peptide wrwycr and the related wrwyrggrywrw entered cancer cells and reduced cell survival in a dose- and time-dependent manner. Early markers for DNA damage, γH2AX foci and 53BP1 foci, increased with dose and/or time exposure to the peptides. DNA breaks persisted at least 48 h, and both checkpoint proteins Chk1 and Chk2 were activated. The passage of the cells from S to G2/M was blocked even after 72 h. Apoptosis, however, was not induced in either HeLa or PC3 cells. Based on colony-forming assays, about 35% peptide-induced cytotoxicity was irreversible. Finally, sublethal doses of peptide wrwycr (50–100 µM) in conjunction with sublethal doses of several DNA damaging agents (etoposide, doxorubicin, and HU) reduced cell survival at least additively and sometimes synergistically. Taken together, the results suggest that the peptides merit further investigation as proof-of-principle molecules for a new class of anti-cancer therapeutics, in particular in combination with other DNA damaging therapies.

Influence of Natural and Synthetic Histone Deacetylase Inhibitors on Chromatin

Histone deacetylase inhibitors (HDACIs) have emerged as a new class of anticancer therapeutics. The hydroxamic acid, suberoylanilide hydroxamic acid (Vorinostat, Zolinza™), and the cyclic peptide, depsipeptide (Romidepsin, Istodax™), were approved by the U.S. Food and Drug Administration (FDA) for the treatment of cutaneous T-cell lymphoma in 2006 and 2009, respectively. At least 15 HDACIs are currently undergoing clinical trials either alone or in combination with other therapeutic modalities for the treatment of numerous hematological and solid malignancies. The potential utility of HDACIs has been extended to nononcologic applications, including autoimmune disorders, inflammation, diseases of the central nervous system, and malaria. Given the promise of HDACIs, there is growing interest in the potential of dietary compounds that possess HDAC inhibition activity. This review is focused on the identification of and recent findings with HDACIs from dietary, medicinal plant, and microbial sources. We discuss the mechanisms of action and clinical potential of natural HDACIs. Apart from identification of further HDACI compounds from dietary sources, further research will be aimed at understanding the effects on gene regulation on lifetime exposure to these compounds. Another important issue that requires clarification.

Histone deacetylase inhibitors augment doxorubicin-induced DNA damage in cardiomyocytes.

Histone deacetylase inhibitors have emerged as a new class of anticancer therapeutics with suberoylanilide hydroxamic acid (Vorinostat) and depsipeptide (Romidepsin) already being approved for clinical use. Numerous studies have identified that histone deacetylase inhibitors will be most effective in the clinic when used in combination with conventional cancer therapies such as ionizing radiation and chemotherapeutic agents. One promising combination, particularly for hematologic malignancies, involves the use of histone deacetylase inhibitors with the anthracycline, doxorubicin. However, we previously identified that trichostatin A can potentiate doxorubicin-induced hypertrophy, the dose-limiting side-effect of the anthracycline, in cardiac myocytes. Here we have the extended the earlier studies and evaluated the effects of combinations of the histone deacetylase inhibitors, trichostatin A, valproic acid and sodium butyrate on doxorubicin-induced DNA double-strand breaks in cardiomyocytes. Using γH2AX as a molecular marker for the DNA lesions, we identified that all of the broad-spectrum histone deacetylase inhibitors tested augment doxorubicin-induced DNA damage. Furthermore, it is evident from the fluorescence photomicrographs of stained nuclei that the histone deacetylase inhibitors also augment doxorubicin-induced hypertrophy. These observations highlight the importance of investigating potential side-effects, in relevant model systems, which may be associated with emerging combination therapies for cancer.

Structure−Activity Study To Develop Cationic Lipid-Conjugated Haloperidol Derivatives as a New Class of Anticancer Therapeutics

Haloperidol (HP), a neuroleptic drug, shows high affinity toward σ receptors (SR). HP and reduced-HP at higher concentration were known to induce apoptosis in SR-overexpressing carcinomas and melanomas. Herein, we report the development of cationic lipid-conjugated haloperidol as a new class of anticancer therapeutics. In comparison to HP, the C-8 carbon chain analogue (HP-C8) showed significantly high, SR-assisted antiproliferative activity against cancer cells via caspase-3-mediated apoptosis and down-regulation of pAkt. Moreover, melanoma tumor aggressiveness in HP-C8-treated mice was significantly lower than that in HP-treated mice. HP-C8 simultaneously reduced Akt-protein level and increased Bax/Bcl-2 ratio in vascular endothelial cells, thereby indicating a possible protein kinase down-regulatory and apoptosis inducing role in tumor-associated vascular cells. In conclusion, we developed σ receptor-targeting cationic lipid-modified HP derivatives as a promising class of anticancer therapeutic that concurrently affects cancer and tumor environment associated angiogenic vascular cells through induction of apoptosis and Akt protein down-regulation.

Trichostatin A accentuates doxorubicin-induced hypertrophy in cardiac myocytes

Histone deacetylase inhibitors represent a new class of anticancer therapeutics and the expectation is that they will be most effective when used in combination with conventional cancer therapies, such as the anthracycline, doxorubicin. The dose-limiting side effect of doxorubicin is severe cardiotoxicity and evaluation of the effects of combinations of the anthracycline with histone deacetylase inhibitors in relevant models is important. We used a well-established in vitro model of doxorubicin-induced hypertrophy to examine the effects of the prototypical histone deacetylase inhibitor, Trichostatin A. Our findings indicate that doxorubicin modulates the expression of the hypertrophy-associated genes, ventricular myosin light chain-2, the alpha isoform of myosin heavy chain and atrial natriuretic peptide, an effect which is augmented by Trichostatin A. Furthermore, we show that Trichostatin A amplifies doxorubicin-induced DNA double strand breaks, as assessed by γH2AX formation. More generally, our findings highlight the importance of investigating potential side effects that may be associated with emerging combination therapies for cancer.

Hexavalent bispecific antibodies represent a new class of anticancer therapeutics: 1. Properties of anti-CD20/CD22 antibodies in lymphoma

AbstractThe dock and lock (DNL) method is a new technology for generating multivalent antibodies. Here, we report in vitro and in vivo characterizations of 20-22 and 22-20, a pair of humanized hexavalent anti-CD20/22 bispecific antibodies (bsAbs) derived from veltuzumab (v-mab) and epratuzumab (e-mab). The 22-20 was made by site-specific conjugation of e-mab to 4 Fabs of v-mab; 20-22 is of the opposite configuration, composing v-mab and 4 Fabs of e-mab. Each bsAb translocates both CD22 and CD20 into lipid rafts, induces apoptosis and growth inhibition without second-antibody crosslinking, and is significantly more potent in killing lymphoma cells in vitro than their parental antibodies. Although both bsAbs triggered antibody-dependent cellular toxicity, neither displayed complement-dependent cytotoxicity. Intriguingly, 22-20 and 20-22 killed human lymphoma cells in preference to normal B cells ex vivo, whereas the parental v-mab depleted malignant and normal B cells equally. In vivo studies in Daudi tumors revealed 20-22, despite having a shorter serum half-life, had antitumor efficacy comparable with equimolar v-mab; 22-20 was less potent than 20-22 but more effective than e-mab and control bsAbs. These results indicate multiple advantages of hexavalent anti-CD20/22 bsAbs over the individual parental antibodies and suggest that these may represent a new class of cancer therapeutics.

Combining PCI-24781, a Novel Histone Deacetylase Inhibitor, with Chemotherapy for the Treatment of Soft Tissue Sarcoma

Histone deactylase inhibitors (HDACi) are a promising new class of anticancer therapeutics; however, little is known about HDACi activity in soft tissue sarcoma (STS), a heterogeneous cohort of mesenchymal origin malignancies. Consequently, we investigated the novel HDACi PCI-24781, alone/in combination with conventional chemotherapy, to determine its potential anti-STS-related effects and the underlying mechanisms involved. Immunoblotting was used to evaluate the effects of PCI-24781 on histone and nonhistone protein acetylation and expression of potential downstream targets. Cell culture-based assays were utilized to assess the effects of PCI-24781 on STS cell growth, cell cycle progression, apoptosis, and chemosensitivity. Quantitative reverse transcription-PCR, chromatin immunoprecipitation, and reporter assays helped elucidate molecular mechanisms resulting in PCI-24781-induced Rad51 repression. The effect of PCI-24781, alone or with chemotherapy, on tumor and metastatic growth was tested in vivo using human STS xenograft models. PCI-24781 exhibited significant anti-STS proliferative activity in vitro, inducing S phase depletion, G(2)/M cell cycle arrest, and increasing apoptosis. Superior effects were seen when combined with chemotherapy. A PCI-24781-induced reduction in Rad51, a major mediator of DNA double-strand break homologous recombination repair, was shown and may be a mechanism underlying PCI-24781 chemosensitization. We showed that PCI-24781 transcriptionally represses Rad51 through an E2F binding-site on the Rad51 proximal promoter. Although single-agent PCI-24781 had modest effects on STS growth and metastasis, marked inhibition was observed when combined with chemotherapy. In light of these findings, this novel molecular-based combination may be applicable to multiple STS histologic subtypes, and potentially merits rigorous evaluation in human STS clinical trials.

Induction of Foxp3 + regulatory T cells with histone deacetylase inhibitors

Histone deacetylase inhibitors are under investigation in the clinic as a new class of anti-cancer therapeutics. While recent studies have also suggested their potential as inhibitors of a wide spectrum of inflammatory reactions, the anti-inflammatory mechanism of action of these compounds is not fully defined. We show here that the histone deacetylase inhibitors MS-275 and SAHA induce the generation of regulatory T cells (Tregs) from anti-CD3/anti-CD28-stimulated human CD4 +CD25 − T cells. These Tregs express the regulatory T cell-associated transcription factor Foxp3 and display suppressive activity against CD4 +CD25 − T cell proliferation. Topical treatment with histone deacetylase inhibitors also induces Foxp3 expression in the draining lymph nodes and the skin in the context of a murine contact hypersensitivity model. These findings suggest that Treg generation may serve as a novel mechanism by which histone deacetylase inhibitors regulate the immune response, and provide an additional rationale for the use of histone deacetylase inhibitors in the treatment of inflammation.