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.

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.

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.

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.

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.

Molecular Modeling of Single- and Double-Hydrocarbon-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 and a subset of acute lymphoblastic leukemias. 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 that 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 CCmut3 inhibitors against Bcr-CC-mediated Bcr-Abl assembly designed to address these constraints through incorporation of all-hydrocarbon staples spanning i and i + 7 positions in α-helix 2 (CPP-CCmut3-st). We utilize computational modeling and biomolecular simulation to evaluate single- and double-stapled CCmut3 candidates in silico for dynamics and binding energetics. We further model a truncated system characterized by the deletion of α-helix 1 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 devoid of the CPP, with a cyclized CPP, and with an open-configuration CPP, for a total of six systems that comprise our library. From this library, we present lead-stapled peptide candidates to be synthesized and evaluated experimentally as our next iteration of inhibitors against Bcr-Abl.

Treatment-Resistant Schizophrenia: Evaluation and Management.

The Psychiatric Consultation Service at Massachusetts General Hospital sees medical and surgical inpatients with comorbid psychiatric symptoms and conditions. During their twice-weekly rounds, Dr Stern and other members of the Consultation Service discuss diagnosis and management of hospitalized patients with complex medical or surgical problems who also demonstrate psychiatric symptoms or conditions. These discussions have given rise to rounds reports that will prove useful for clinicians practicing at the interface of medicine and psychiatry.Prim Care Companion CNS Disord 2024;26(4):23f03692. Author affiliations are listed at the end of this article.

Clozapine: Its Use and Monitoring.

The Psychiatric Consultation Service at Massachusetts General Hospital sees medical and surgical inpatients with comorbid psychiatric symptoms and conditions. During their twice-weekly rounds, Dr Stern and other members of the Consultation Service discuss diagnosis and management of hospitalized patients with complex medical or surgical problems who also demonstrate psychiatric symptoms or conditions. These discussions have given rise to rounds reports that will prove useful for clinicians practicing at the interface of medicine and psychiatry.Prim Care Companion CNS Disord 2024;26(4):23f03702. Author affiliations are listed at the end of this article.

Disrupting BCR-ABL in combination with secondary leukemia-specific pathways in CML cells leads to enhanced apoptosis and decreased proliferation.

Chronic myeloid leukemia (CML) is a myeloproliferative disorder caused by expression of the fusion gene BCR-ABL following a chromosomal translocation in the hematopoietic stem cell. Therapeutic management of CML uses tyrosine kinase inhibitors (TKIs), which block ABL-signaling and effectively kill peripheral cells with BCR-ABL. However, TKIs are not curative, and chronic use is required in order to treat CML. The primary failure for TKIs is through the development of a resistant population due to mutations in the TKI binding regions. This led us to develop the mutant coiled-coil, CC(mut2), an alternative method for BCR-ABL signaling inhibition by targeting the N-terminal oligomerization domain of BCR, necessary for ABL activation. In this article, we explore additional pathways that are important for leukemic stem cell survival in K562 cells. Using a candidate-based approach, we test the combination of CC(mut2) and inhibitors of unique secondary pathways in leukemic cells. Transformative potential was reduced following silencing of the leukemic stem cell factor Alox5 by RNA interference. Furthermore, blockade of the oncogenic protein MUC-1 by the novel peptide GO-201 yielded reductions in proliferation and increased cell death. Finally, we found that inhibiting macroautophagy using chloroquine in addition to blocking BCR-ABL signaling with the CC(mut2) was most effective in limiting cell survival and proliferation. This study has elucidated possible combination therapies for CML using novel blockade of BCR-ABL and secondary leukemia-specific pathways.