Computational Modeling of Stapled Coiled-Coil Inhibitors Against Bcr-Abl: Toward a Treatment Strategy for CML.

The chimeric oncoprotein Bcr-Abl is the causative agent of virtually all chronic myeloid leukemias (CML) and a subset of acute lymphoblastic leukemias (ALL). As a result of the so-called Philadelphia Chromosome translocation t(9;22), Bcr-Abl manifests as a constitutively active tyrosine kinase which promotes leukemogenesis by activation of cell cycle signaling pathways. Constitutive and oncogenic activation is mediated by an N-terminal coiled-coil oligomerization domain in Bcr (Bcr-CC), presenting a therapeutic target for inhibition of Bcr-Abl activity toward the treatment of Bcr-Abl+ leukemias. Previously, we demonstrated that a rationally designed Bcr-CC mutant, CCmut3, exerts a dominant negative effect upon Bcr-Abl activity by preferential oligomerization with Bcr-CC. Moreover, we have shown conjugation to a leukemia-specific cell-penetrating peptide (CPP-CCmut3) improves intracellular delivery and activity. However, our full-length CPP-CCmut3 construct (81 aa) is encumbered by an intrinsically high degree of conformational variability and susceptibility to proteolytic degradation, relative to traditional small molecule therapeutics. Here, we iterate a new generation of our inhibitor against Bcr-CC mediated Bcr-Abl assembly that is designed to address these constraints through incorporation of all-hydrocarbon staples spanning i, i + 7 positions in helix α2 (CPP-CCmut3-st). We utilize computational modeling and biomolecular simulation to design and characterize single and double staple candidates in silico, evaluating binding energetics and building upon our seminal work modeling single hydrocarbon staples when applied to a truncated Bcr-CC sequence. This strategy enables us to efficiently build, characterize, and screen lead single/double stapled CPP-CCmut3-st candidates for experimental studies and validation in vitro and in vivo. In addition to full-length CPP-CCmut, we model a truncated system characterized by deletion of helix α1 and the flexible-loop linker, which are known to impart high conformational variability. To study the impact of the N-terminal cyclic CPP toward model stability and inhibitor activity, we also model the full-length and truncated systems without CPP, with cyclized CPP, and with linear CPP, for a total of six systems which comprise our library. From this library, we present lead stapled peptide candidates to be synthesized and evaluated experimentally as our next-generation inhibitors against Bcr-Abl.

Systematic single amino acid affinity tuning of CD229 CAR T cells retains efficacy against multiple myeloma and eliminates on-target off-tumor toxicity.

T cells expressing chimeric antigen receptors (CARs) have shown remarkable therapeutic activity against different types of cancer. However, the wider use of CAR T cells has been hindered by the potential for life-threatening toxicities due to on-target off-tumor killing of cells expressing low amounts of the target antigen. CD229, a signaling lymphocyte-activation molecule (SLAM) family member, has previously been identified as a target for CAR T cell-mediated treatment of multiple myeloma (MM) due to its high expression on the surfaces of MM cells. CD229 CAR T cells have shown effective clearance of MM cells in vitro and in vivo. However, healthy lymphocytes also express CD229, albeit at lower amounts than MM cells, causing their unintended targeting by CD229 CAR T cells. To increase the selectivity of CD229 CAR T cells for MM cells, we used a single amino acid substitution approach of the CAR binding domain to reduce CAR affinity. To identify CARs with increased selectivity, we screened variant binding domains using solid-phase binding assays and biolayer interferometry and determined the cytotoxic activity of variant CAR T cells against MM cells and healthy lymphocytes. We identified a CD229 CAR binding domain with micromolar affinity that, when combined with overexpression of c-Jun, confers antitumor activity comparable to parental CD229 CAR T cells but lacks the parental cells' cytotoxic activity toward healthy lymphocytes in vitro and in vivo. The results represent a promising strategy to improve the efficacy and safety of CAR T cell therapy that requires clinical validation.

Mapping the Evidence for Opioid-Mediated Changes in Malignancy and Chemotherapeutic Efficacy: Protocol for a Scoping Review.

BACKGROUND: Numerous reports contend opioids can augment or inhibit malignancy. At present, there is no consensus on the risk or benefit posed by opioids on malignancy or chemotherapeutic activity. Distinguishing the consequences of opioid use from pain and its management is challenging. Additionally, opioid concentration data is often lacking in clinical studies. A scoping review approach inclusive of preclinical and clinical data will improve our understanding of the risk-benefit relationship concerning commonly prescribed opioids and cancer and cancer treatment. OBJECTIVE: The aim of the study is to map diverse studies spanning from preclinical to clinical regarding opioids with malignancy and its treatment. METHODS: This scoping review will use the Arksey six stages framework to (1) identify the research question; (2) identify relevant studies; (3) select studies meeting criteria; (4) extract and chart data; (5) collate, summarize, and report results; and (6) conduct expert consultation. An initial pilot study was undertaken to (1) parameterize the extent and scale of existing data for an evidence review, (2) identify key factors to be extracted in systematic charting efforts, and (3) assess opioid concentration as a variable for its relevance to the central hypothesis. Six databases will be searched with no filters: MEDLINE, Embase, CINAHL Complete, Cochrane Library, Biological Sciences Collection, and International Pharmaceutical Abstracts. Trial registries will include ClinicalTrials.gov, Cochrane CENTRAL, International Standard Randomised Controlled Trial Number Registry, European Union Clinical Trials Register, and World Health Organization International Clinical Trials Registry. Eligibility criteria will include preclinical and clinical study data on opioids effects on tumor growth or survival, or alteration on the antineoplastic activity of chemotherapeutics. We will chart data on (1) opioid concentration from human subjects with cancer, yielding a "physiologic range" to better interpret available preclinical data; (2) patterns of opioid exposure with disease and treatment-related patient outcomes; and (3) the influence of opioids on cancer cell survival, as well as opioid-related changes to cancer cell susceptibility for chemotherapeutics. RESULTS: This scoping review will present results in narrative forms as well as with the use of tables and diagrams. Initiated in February 2021 at the University of Utah, this protocol is anticipated to generate a scoping review by August 2023. The results of the scoping review will be disseminated through scientific conference proceedings and presentations, stakeholder meetings, and by publication in a peer-reviewed journal. CONCLUSIONS: The findings of this scoping review will provide a comprehensive description of the consequences of prescription opioids on malignancy and its treatment. By incorporating preclinical and clinical data, this scoping review will invite novel comparisons across study types that could inform new basic, translational, and clinical studies regarding risks and benefits of opioid use among patients with cancer. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): PRR1-10.2196/38167.

The Challenges and Prospects of p53-Based Therapies in Ovarian Cancer.

It has been well established that mutations in the tumor suppressor gene, p53, occur readily in a vast majority of cancer tumors, including ovarian cancer. Typically diagnosed in stages three or four, ovarian cancer is the fifth leading cause of death in women, despite accounting for only 2.5% of all female malignancies. The overall 5-year survival rate for ovarian cancer is around 47%; however, this drops to an abysmal 29% for the most common type of ovarian cancer, high-grade serous ovarian carcinoma (HGSOC). HGSOC has upwards of 96% of cases expressing mutations in p53. Therefore, wild-type (WT) p53 and p53-based therapies have been explored as treatment options via a plethora of drug delivery vehicles including nanoparticles, viruses, polymers, and liposomes. However, previous p53 therapeutics have faced many challenges, which have resulted in their limited translational success to date. This review highlights a selection of these historical p53-targeted therapeutics for ovarian cancer, why they failed, and what the future could hold for a new generation of this class of therapies.

p53-Bad* Fusion Gene Therapy Induces Apoptosis In Vitro and Reduces Zebrafish Tumor Burden in Hepatocellular Carcinoma.

With few curative treatments and a global yearly death rate of over 800,000, hepatocellular carcinoma (HCC) desperately needs new therapies. Although wild-type p53 gene therapy has been shown to be safe in HCC patients, it has not shown enough efficacy to merit approval. This work aims to show how p53 can be re-engineered through fusion to the pro-apoptotic BH3 protein Bcl-2 antagonist of cell death (Bad) to improve anti-HCC activity and potentially lead to a novel HCC therapeutic, p53-Bad*. p53-Bad* is a fusion of p53 and Bad, with two mutations, S112A and S136A. We determined mitochondrial localization of p53-Bad* in liver cancer cell lines with varying p53 mutation statuses via fluorescence microscopy. We defined the apoptotic activity of p53-Bad* in four liver cancer cell lines using flow cytometry. To determine the effects of p53-Bad* in vivo, we generated and analyzed transgenic zebrafish expressing hepatocyte-specific p53-Bad*. p53-Bad* localized to the mitochondria regardless of the p53 mutation status and demonstrated superior apoptotic activity over WT p53 in early, middle, and late apoptosis assays. Tumor burden in zebrafish HCC was reduced by p53-Bad* as measured by the liver-to-body mass ratio and histopathology. p53-Bad* induced significant apoptosis in zebrafish HCC as measured by TUNEL staining but did not induce apoptosis in non-HCC fish. p53-Bad* can induce apoptosis in a panel of liver cancer cell lines with varying p53 mutation statuses and induce apoptosis/reduce HCC tumor burden in vivo in zebrafish. p53-Bad* warrants further investigation as a potential new HCC therapeutic.

Roadmap to affinity-tuned antibodies for enhanced chimeric antigen receptor T cell function and selectivity.

Chimeric antigen receptor (CAR) T cells have revolutionized cancer treatment. CARs use antibody-derived binding domains to redirect T cells to antigens expressed on the surface of cancer cells. However, the high affinity of most currently used CAR-binding domains results in excessive T-cell activation limiting CAR T-cell persistence and the inability to differentiate between antigen-high tumor cells and antigen-low healthy cells. We review recent data on the use of low-affinity CAR-binding domains and evaluate technologies and approaches to engineer and screen low-affinity antibody variants for CAR T-cell development. We propose an ideal workflow for the generation of optimal low-affinity binders derived from existing antibodies to streamline the development of more functional and selective therapeutics.

Advances in delivery vectors for gene therapy in liver cancer.

Hepatocellular carcinoma (HCC) is the third most common cause of cancer death globally, mainly due to lack of effective treatments - a problem that gene therapy is poised to solve. Successful gene therapy requires safe and efficient delivery vectors, and recent advances in both viral and nonviral vectors have made an important impact on HCC gene therapy delivery. This review explores how adenoviral, retroviral and adeno-associated viral vectors have been modified to increase safety and delivery capacity, highlighting studies and clinical trials using these vectors for HCC gene therapy. Nanoparticles, liposomes, exosomes and virosomes are also featured in their roles as HCC gene delivery vectors. Finally, new discoveries in gene editing technology and their impacts on HCC gene therapy are discussed.

p53-Bad: A Novel Tumor Suppressor/Proapoptotic Factor Hybrid Directed to the Mitochondria for Ovarian Cancer Gene Therapy.

Clinical trials involving p53 gene therapy for ovarian cancer failed due to the dominant negative inhibition of wild-type p53 and multiple genetic aberrations in ovarian cancer. To overcome this problem, we have designed a more potent chimeric gene fusion, called p53-Bad, that combines p53 with the mitochondrial pro-apoptotic factor Bad. Unlike wild-type p53, which acts as a nuclear transcription factor, this novel p53-Bad construct has multiple unique mechanisms of action including a direct and rapid apoptotic effect at the mitochondria. The mitochondrial localization, transcription activity, and apoptotic activity of the constructs were tested. The results suggest that p53 can be effectively targeted to the mitochondria by controlling the phosphorylation of pro-apoptotic Bad, which can only localize to the mitochondria when Ser-112 and Ser-136 of Bad are unphosphorylated. By introducing S112A and S136A mutations, p53-Bad fusion cannot be phosphorylated at these two sites and always localizes to the mitochondria. p53-Bad constructs also have superior activity over p53 and Bad alone. The apoptotic activity is consistent in many ovarian cancer cell lines regardless of the endogenous p53 status. Both p53 and the BH3 domain of Bad contribute to the superior activity of p53-Bad. Our data suggests that p53-Bad fusions are capable of inducing apoptosis and should be further pursued for gene therapy for ovarian cancer.

Narrowing the field: cancer-specific promoters for mitochondrially-targeted p53-BH3 fusion gene therapy in ovarian cancer.

BACKGROUND: Despite years of research, the treatment options and mortality rate for ovarian cancer remain relatively stagnant. Resistance to chemotherapy and high heterogeneity in mutations contribute to ovarian cancer's lethality, including many mutations in tumor suppressor p53. Though wild type p53 gene therapy clinical trials failed in ovarian cancer, mitochondrially-targeted p53 fusion constructs, including a fusion with pro-apoptotic protein Bad, have shown much higher apoptotic potential than wild type p53 in vitro. Due to the inherent toxicities of mitochondrial apoptosis, cancer-specificity for the p53 fusion constructs must be developed. Cancer-specific promoters such as hTERT, hTC, Brms1, and Ran have shown promise in ovarian cancer. RESULTS: Of five different lengths of hTERT promoter, the - 279/+ 5 length relative to the transcription start site showed the highest activity across a panel of ovarian cancer cells. In addition to - 279/+ 5, promoters hTC (an hTERT/CMV promoter hybrid), Brms1, and Ran were tested as drivers of mitochondrially-targeted p53-Bad and p53-Bad* fusion gene therapy constructs. p53-Bad* displayed cancer-specific killing in all ovarian cancer cell lines when driven by hTC, - 279/+ 5, or Brms1. CONCLUSIONS: Cancer-specific promoters hTC, - 279/+ 5, and Brms1 all display promise in driving p53-Bad* gene therapy for treatment of ovarian cancer and should be moved forward into in vivo studies. -279/+ 5 displays lower expression levels in fewer cells, but greater cancer specificity, rendering it most useful for gene therapeutics with high toxicity to normal cells. hTC and Brms1 show higher transfection and expression levels with some cancer specificity, making them ideal for lowering toxicity in order to increase dose without as much of a reduction in the number of cancer cells expressing the gene construct. Having a variety of promoters available means that patient genetic testing can aid in choosing a promoter, thereby increasing cancer-specificity and giving patients with ovarian cancer a greater chance at survival.

Mitochondrially targeted p53 or DBD subdomain is superior to wild type p53 in ovarian cancer cells even with strong dominant negative mutant p53.

BACKGROUND: While tumor suppressor p53 functions primarily as a transcription factor in the nucleus, cellular stress can cause p53 to translocate to the mitochondria and directly trigger a rapid apoptotic response. We have previously shown that fusing p53 (or its DNA binding domain, DBD, alone) to the mitochondrial targeting signal (MTS) from Bak or Bax can target p53 to the mitochondria and induce apoptosis in gynecological cancer cell lines including cervical cancer cells (HeLa; wt p53), ovarian cancer cells (SKOV-3; p53 267del non-expressing), and breast cancer cells (T47D; L194F p53 mutation). However, p53 with Bak or Bax MTSs have not been previously tested in cancers with strong dominant negative (DN) mutant p53 which are capable of inactivating wt p53 by homo-oligomerization. Since p53-Bak or Bax MTS constructs act as monomers, they are not subject to DN inhibition. For this study, the utility of p53-Bak or p53-Bax MTS constructs was tested for ovarian cancers which are known to have varying p53 statuses, including a strong DN contact mutant p53 (Ovcar-3 cells), a p53 DN structural mutant (Kuramochi cells), and a p53 wild type, low expressing cells (ID8). RESULTS: Our mitochondrial p53 constructs were tested for their ability to localize to the mitochondria in both mutant non-expressing p53 (Skov-3) and p53 structural mutant (Kuramochi) cell lines using fluorescence microscopy and a nuclear transcriptional activity assay. The apoptotic activity of these mitochondrial constructs was determined using a mitochondrial outer membrane depolarization assay (TMRE), caspase assay, and a late stage cell death assay (7-AAD). We also tested the possibility of using our constructs with paclitaxel, the current standard of care in ovarian cancer treatment. Our data indicates that our mitochondrial p53 constructs are able to effectively localize to the mitochondria in cancer cells with structural mutant p53 and induce apoptosis in many ovarian cancer cell lines with different p53 statuses. These constructs can also be used in combination with paclitaxel for an increased apoptotic effect. CONCLUSIONS: The results suggest that targeting p53 to mitochondria can be a new strategy for ovarian cancer treatment.

Computational Modeling of Stapled Peptides toward a Treatment Strategy for CML and Broader Implications in the Design of Lengthy Peptide Therapeutics.

The oncogenic gene product Bcr-Abl is the principal cause of chronic myeloid leukemia, and although several therapies exist to curb the aberrant kinase activity of Bcr-Abl through targeting of the Abl kinase domain, these therapies are rendered ineffective by frequent mutations in the corresponding gene. It has been demonstrated that a designed protein, known as CCmut3, is able to produce a dominant negative inactivating effect on Bcr-Abl kinase by preferentially oligomerizing with the N-terminal coiled-coil oligomerization domain of Bcr-Abl (Bcr-CC) to effectively reduce the oncogenic potential of Bcr-Abl. However, the sheer length of the CCmut3 peptide introduces a high degree of conformational variability and opportunity for targeting by intracellular proteolytic mechanisms. Here, we have examined the effects of introducing one or two molecular staples, or cross-links, spanning i, i + 7 backbone residues of the CCmut3 construct, which have been suggested to reinforce α-helical conformation, enhance cellular internalization, and increase resistance to proteolytic degradation, leading to enhanced pharmacokinetic properties. The importance of optimizing staple location along a highly tuned biological construct such as CCmut3 has been widely emphasized and, as such, we have employed in silico techniques to swiftly build, relax, and characterize a large number of candidates. This approach effectively allowed exploring each and every possible staple location along the peptide backbone so that every possible candidate is considered. Although many of the stapled candidate peptides displayed enhanced binding characteristics for Bcr-CC and improved conformational stability in the (Bcr-CC) bound form, simulations of the stapled peptides in the unbound form revealed widespread conformational variability among stapled candidates dependent on staple type and location, implicating the molecular replacement of helix-stabilizing residues with staple-containing residues in disrupting the native α-helical conformation of CCmut3, further highlighting a need for careful optimization of the CCmut3 construct. A candidate set has been assembled, which retains the native backbone α-helical integrity in both the bound and unbound forms while providing enhanced binding affinity for the Bcr-CC target, as research disseminated in this manuscript is intended to guide the development of a next-generation CCmut3 inhibitor peptide in an experimental setting.

Application of Thiol-yne/Thiol-ene Reactions for Peptide and Protein Macrocyclizations.

The application of thiol-yne/thiol-ene reactions to synthesize mono- and bicyclic-stapled peptides and proteins is reported. First, a thiol-ene-based peptide-stapling method in aqueous conditions was developed. This method enabled the efficient stapling of recombinantly expressed coil-coiled proteins. The resulting stapled protein demonstrated higher stability in its secondary structure than the unstapled version. Furthermore, a thiol-yne coupling was performed by using an α,ω-diyne to react with two cysteine residues to synthesize a stapled peptide with two vinyl sulfide groups. The stapled peptide could further react with another biscysteine peptide to yield a bicyclic stapled peptide with enhanced properties. For example, the cell permeability of a stapled peptide was further increased by appending an oligoarginine cell-penetrating peptide. The robustness and versatility of thiol-yne/thiol-ene reactions that can be applied to both synthetic and expressed peptides and proteins were demonstrated.

Delivery of drugs and macromolecules to the mitochondria for cancer therapy.

Mitochondria are organelles that have pivotal functions in producing the energy necessary for life and executing the cell death pathway. Targeting drugs and macromolecules to the mitochondria may provide an effective means of inducing cell death for cancer therapy, and has been actively pursued in the last decade. This review will provide a brief overview of mitochondrial structure and function, how it relates to cancer, and importantly, will discuss different strategies of mitochondrial delivery including delivery using small molecules, peptides, genes encoding proteins and MTSs, and targeting polymers/nanoparticles with payloads to the mitochondria. The advantages and disadvantages for each strategy will be discussed. Specific examples using the latest strategies for mitochondrial targeting will be evaluated, as well as potential opportunities for specific mitochondrial compartment localization, which may lead to improvements in mitochondrial therapeutics. Future perspectives in mitochondrial targeting of drugs and macromolecules will be discussed. Currently this is an under-explored area that is prime for new discoveries in cancer therapeutics.

Inhibition of bcr-abl in human leukemic cells with a coiled-coil protein delivered by a leukemia-specific cell-penetrating Peptide.

The oncoprotein Bcr-Abl is the cause of chronic myeloid leukemia (CML).1 Current therapies target the tyrosine kinase domain of Bcr-Abl, but resistance to these drugs is common.2 Bcr-Abl homo-oligomerization via its N-terminal coiled-coil (CC) domain is required for tyrosine kinase activity.3 Our previous work has shown that it is possible to inhibit Bcr-Abl activity by targeting the CC domain with a peptidomemetic known as CC(mut3), delivered as a plasmid.4 In this study, CC(mut3) is delivered to cells as a protein by utilizing a leukemia-specific cell-penetrating peptide (CPP).5 Here, recombinant CPP-CC(mut3) was expressed, purified, and tested for its antioncogenic activity. CPP-CC(mut3) was able to enter two leukemic cell lines (K562 and Ba/F3-P210) and inhibit Bcr-Abl activity as shown by induction of necrosis/apoptosis via 7-AAD/Annexin V staining, reduction of oncogenic potential in colony forming assays, reduction of cell proliferation, and inhibition of Bcr-Abl phosphorylation (kinase activity). Further, CPP-CC(mut3) did not enter nonleukemic cell lines (HEK293 and MCF-7). While CPP-CC(mut3) was able to enter the parental, nonleukemic Bcr-Abl(-) Ba/F3 pro-B cell line, it revealed no signs of activity in the assays performed, as expected. These results indicate the feasibility of using CPP-CC(mut3) as a therapeutic against CML.

Resistant mutations in CML and Ph(+)ALL - role of ponatinib.

In 2012, ponatinib (Iclusig(®)), an orally available pan-BCR-ABL tyrosine kinase inhibitor (TKI) developed by ARIAD Pharmaceuticals, Inc., was approved by the US Food and Drug Administration for use in resistant or intolerant chronic myeloid leukemia (CML) and Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph(+)ALL). Ponatinib is the only approved TKI capable of inhibiting BCR-ABL with the gatekeeper T315I kinase domain mutation, known to be the cause for 20% of resistant or relapsed CML cases. In 2013, ponatinib sales were temporarily suspended due to serious side effects seen in nearly 12% of the patient population. These side effects are thought to stem from the potent nature and pan-activity of this TKI. ARIAD Pharmaceuticals, Inc. has since been permitted to resume sales and marketing of ponatinib to a limited patient population with an expanded black box warning. In the following review, the use of ponatinib in CML and Ph(+)ALL will be discussed. Mechanisms of resistance in CML are discussed, which provide insight and background into the need for this third generation TKI, followed by the molecular design and pharmacology of ponatinib, which lead to its success as a therapeutic. Finally, the efficacy, safety, and tolerability of ponatinib will be highlighted, including summaries of the important clinical trials involving ponatinib as well as its current place in therapy.

Re-engineered p53 chimera with enhanced homo-oligomerization that maintains tumor suppressor activity.

The use of the tumor suppressor p53 for gene therapy of cancer is limited by the dominant negative inactivating effect of mutant endogenous p53 in cancer cells. We have shown previously that swapping the tetramerization domain (TD) of p53 with the coiled-coil (CC) from Bcr allows for our chimeric p53 (p53-CC) to evade hetero-oligomerization with endogenous mutant p53. This enhances the utility of this construct, p53-CC, for cancer gene therapy. Because domain swapping to create p53-CC could result in p53-CC interacting with endogenous Bcr, which is ubiquitous in cells, modifications on the CC domain are necessary to minimize potential interactions with Bcr. Hence, we investigated the possible design of mutations that will improve homodimerization of CC mutants and disfavor hetero-oligomerization with wild-type CC (CCwt), with the goal of minimizing potential interactions with endogenous Bcr in cells. This involved integrated computational and experimental approaches to rationally design an enhanced version of our chimeric p53-CC tumor suppressor. Indeed, the resulting lead candidate p53-CCmutE34K-R55E avoids binding to endogenous Bcr and retains p53 tumor suppressor activity. Specifically, p53-CCmutE34K-R55E exhibits potent apoptotic activity in a variety of cancer cell lines, regardless of p53 status (in cells with mutant p53, wild-type p53, or p53-null cells). This construct overcomes the dominant negative effect limitation of wt p53 and has high significance for future gene therapy for treatment of cancers characterized by p53 dysfunction, which represent over half of all human cancers.

Delivery of a monomeric p53 subdomain with mitochondrial targeting signals from pro-apoptotic Bak or Bax.

PURPOSE: p53 targeted to the mitochondria is the fastest and most direct pathway for executing p53 death signaling. The purpose of this work was to determine if mitochondrial targeting signals (MTSs) from pro-apoptotic Bak and Bax are capable of targeting p53 to the mitochondria and inducing rapid apoptosis. METHODS: p53 and its DNA-binding domain (DBD) were fused to MTSs from Bak (p53-BakMTS, DBD-BakMTS) or Bax (p53-BaxMTS, DBD-BaxMTS). Mitochondrial localization was tested via fluorescence microscopy in 1471.1 cells, and apoptosis was detected via 7-AAD in breast (T47D), non-small cell lung (H1373), ovarian (SKOV-3) and cervical (HeLa) cancer cells. To determine that apoptosis is via the intrinsic apoptotic pathway, TMRE and caspase-9 assays were conducted. Finally, the involvement of p53/Bak specific pathway was tested. RESULTS: MTSs from Bak and Bax are capable of targeting p53 to the mitochondria, and p53-BakMTS and p53-BaxMTS cause apoptosis through the intrinsic apoptotic pathway. Additionally, p53-BakMTS, DBD-BakMTS, p53-BaxMTS and DBD-BaxMTS caused apoptosis in T47D, H1373, SKOV-3 and HeLa cells. The apoptotic mechanism of p53-BakMTS and DBD-BakMTS was Bak dependent. CONCLUSION: Our data demonstrates that p53-BakMTS (or BaxMTS) and DBD-BakMTS (or BaxMTS) cause apoptosis at the mitochondria and can be used as a potential gene therapeutic in cancer.

Basics and recent advances in peptide and protein drug delivery.

While the peptide and protein therapeutic market has developed significantly in the past decades, delivery has limited their use. Although oral delivery is preferred, most are currently delivered intravenously or subcutaneously due to degradation and limited absorption in the gastrointestinal tract. Therefore, absorption enhancers, enzyme inhibitors, carrier systems and stability enhancers are being studied to facilitate oral peptide delivery. Additionally, transdermal peptide delivery avoids the issues of the gastrointestinal tract, but also faces absorption limitations. Due to proteases, opsonization and agglutination, free peptides are not systemically stable without modifications. This review discusses oral and transdermal peptide drug delivery, focusing on barriers and solutions to absorption and stability issues. Methods to increase systemic stability and site-specific delivery are also discussed.

Multidomain targeting of Bcr-Abl by disruption of oligomerization and tyrosine kinase inhibition: toward eradication of CML.

The oncoprotein Bcr-Abl, the causative agent of chronic myeloid leukemia (CML), requires homo-oligomerization via a coiled-coil domain to function [Bartram, C. R.; et al. Nature 1983, 306 (5940), 277-280; and Zhao, X.; et al. Nat. Struct. Biol. 2002, 9(2), 117-120]. While tyrosine kinase inhibitors (TKIs) have shown great efficacy as treatment options for CML, their use may cause an acquisition of mutations in the tyrosine kinase domain, which prevent TKI binding and lead to a loss in activity [Woessner, D. W.; et al. Cancer J. 2011, 17(6), 477-486]. Previously, we have shown that a rationally modified coiled-coil domain (CC(mut3)) can disrupt this oligomerization, inhibit proliferation, and induce apoptosis in CML cells [Dixon, A. S.; et al. Mol. Pharmaceutics 2012, 9(1), 187-195]. Here, we show that using the most recently approved TKI, ponatinib (Iclusig), in combination with CC(mut3) allows a dose reduction of ponatinib and increased therapeutic efficacy in vitro measured by reduction in kinase activity, induction of apoptosis via caspase-3/7 and 7-AAD/Annexin V assays, and reduced transformative ability measured by a colony forming assay. The combination was effective not only in cells containing wild-type Bcr-Abl (K562, Ba/F3-p210) but also cells with Bcr-Abl containing the T315I mutation (Ba/F3-p210-T315I). In addition, we report for the first time the ability of CC(mut3) alone to inhibit the T315I mutant form of Bcr-Abl. This novel combination may prove to be more potent than single agent therapies and should be further explored for clinical use.

A chimeric p53 evades mutant p53 transdominant inhibition in cancer cells.

Because of the dominant negative effect of mutant p53, there has been limited success with wild-type (wt) p53 cancer gene therapy. Therefore, an alternative oligomerization domain for p53 was investigated to enhance the utility of p53 for gene therapy. The tetramerization domain of p53 was substituted with the coiled-coil (CC) domain from Bcr (breakpoint cluster region). Our p53 variant (p53-CC) maintains proper nuclear localization in breast cancer cells detected via fluorescence microscopy and shows a similar expression profile of p53 target genes as wt-p53. Additionally, similar tumor suppressor activities of p53-CC and wt-p53 were detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), annexin-V, 7-aminoactinomycin D (7-AAD), and colony-forming assays. Furthermore, p53-CC was found to cause apoptosis in four different cancer cell lines, regardless of endogenous p53 status. Interestingly, the transcriptional activity of p53-CC was higher than wt-p53 in 3 different reporter gene assays. We hypothesized that the higher transcriptional activity of p53-CC over wt-p53 was due to the sequestration of wt-p53 by endogenous mutant p53 found in cancer cells. Co-immunoprecipitation revealed that wt-p53 does indeed interact with endogenous mutant p53 via its tetramerization domain, while p53-CC escapes this interaction. Therefore, we investigated the impact of the presence of a transdominant mutant p53 on tumor suppressor activities of wt-p53 and p53-CC. Overexpression of a potent mutant p53 along with wt-p53 or p53-CC revealed that, unlike wt-p53, p53-CC retains the same level of tumor suppressor activity. Finally, viral transduction of wt-p53 and p53-CC into a breast cancer cell line that harbors a tumor derived transdominant mutant p53 validated that p53-CC indeed evades sequestration and consequent transdominant inhibition by endogenous mutant p53.