Inhibitors For Cancer Therapy Research Articles

Rational design, synthesis and biological evaluation of dual PARP-1/2 and TNKS1/2 inhibitors for cancer therapy

Poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors are the first and most successful drugs designed to exploit the concept of synthetic lethality (SL) between PARP-1 and BRCA1/2, which provides a novel strategy for tumor treatment. However, narrowed indications and resistance to PARP-1 inhibitors have hampered their further clinical application. Inducing “BRCAness” by targeting other targets, which will directly or indirectly disturb the homologous recombination (HR) repair pathway of double-strand DNA breaks (DSBs), is a promising strategy for expanding the clinical application of PARP-1 inhibitors and overcoming resistance to these inhibitors. Tankyrase1/2 (TNKS1/2) are involved in the nonhomologous end-joining (NHEJ) DNA repair pathway by regulating Wnt/β-catenin signaling. TNKS1/2 can also induce a “BRCAness” phenotype by regulating Wnt signaling, which increases the sensitivity of tumor cells with BRCA proficiency to PARP-1 inhibitors. These results suggest that cotargeting PARP1/2 and TNKS1/2 not only exerts a synergistic effect in the treatment of tumors but also provides a novel strategy for expanding the clinical application of PARP-1 inhibitors and overcoming resistance to PARP-1 inhibitors. Therefore, a series of dual PARP-1/2 and TNKS1/2 inhibitors were rationally designed, synthesized, and evaluated for their pharmacological properties. Among these candidates, compound I-9 showed excellent inhibitory activity as it inhibited PARP-1/2 and TNKS1/2 with IC50 values of 0.25 nM, 1.2 nM, 13.5 nM and 4.15 nM, respectively. I-9 exhibited favorable synergistic antitumor efficacy in both BRCA-mutant and BRCA-wild-type cancer lines. Moreover, I-9 exerted prominent dose-dependent antitumor activity in an HCT116 cell-derived xenograft model and was significantly more efficacious than olaparib and E7449. Overall, the present study indicated that I-9, a dual PARP-1/2 and TNKS1/2 inhibitor, is a novel and promising agent for cancer therapy.

In silico, synthesis and anticancer evaluation of benzamide tryptamine derivatives as novel eEF2K inhibitors

Eukaryotic elongation factor 2 kinase (eEF2K), a member of the atypical α-kinase family, is highly expressed in a variety of tumor tissues. Inhibition of eEF2K function can effectively kill cancer cells without affecting the function of normal cells. Therefore, eEF2K is a promising new target for cancer therapy. In this study, a series of benzamide tryptamine derivatives were designed and synthesized as novel eEF2K inhibitors. The druggability properties of the synthesized compounds were predicted in silico and performed well. The MTT assay indicated that most of these compounds displayed good antiproliferative activity against human leukemia CCRF-CEM and K562 cell lines. The structure–activity relationship (SAR) revealed that substituents with different electronic effects on the C5 position of indole ring or C2′, C4′ positions of benzene ring have a great influence on the anti-proliferative activity. Among them, 5j demonstrated the highest anti-proliferative activity with IC50 value of 1.63–3.54 μM. this compound displayed an effective eEF2K inhibition by down-regulated the level of phosphorylated eEF2 in CCRF-CEM cells. Additionally, the western blot analysis further revealed that 5j also significantly affected eEF2K-related signaling pathways. Anticancer mechanism studies have shown that 5j arrested the cell cycle in G0/G1 and induced CCRF-CEM cells apoptosis. Furthermore, 5j activated cleaved caspase-9, 8, 3 and cleaved PARP in a time-dependent manner, which suggesting that 5j induced cancer cells apoptosis through both intrinsic and extrinsic pathways. In summary, benzamide tryptamine derivative 5j represents a novel and promising lead structure for the development of eEF2K inhibitors in cancer therapy.

Immune Checkpoint Inhibitors in Cancer Therapy.

The discovery of immune checkpoint proteins such as PD-1/PDL-1 and CTLA-4 represents a significant breakthrough in the field of cancer immunotherapy. Therefore, humanized monoclonal antibodies, targeting these immune checkpoint proteins have been utilized successfully in patients with metastatic melanoma, renal cell carcinoma, head and neck cancers and non-small lung cancer. The US FDA has successfully approved three different categories of immune checkpoint inhibitors (ICIs) such as PD-1 inhibitors (Nivolumab, Pembrolizumab, and Cemiplimab), PDL-1 inhibitors (Atezolimumab, Durvalumab and Avelumab), and CTLA-4 inhibitor (Ipilimumab). Unfortunately, not all patients respond favourably to these drugs, highlighting the role of biomarkers such as Tumour mutation burden (TMB), PDL-1 expression, microbiome, hypoxia, interferon-γ, and ECM in predicting responses to ICIs-based immunotherapy. The current study aims to review the literature and updates on ICIs in cancer therapy.

Distinct molecular and immune hallmarks of inflammatory arthritis induced by immune checkpoint inhibitors for cancer therapy

Immune checkpoint inhibitors are associated with immune-related adverse events (irAEs), including arthritis (arthritis-irAE). Management of arthritis-irAE is challenging because immunomodulatory therapy for arthritis should not impede antitumor immunity. Understanding of the mechanisms of arthritis-irAE is critical to overcome this challenge, but the pathophysiology remains unknown. Here, we comprehensively analyze peripheral blood and/or synovial fluid samples from 20 patients with arthritis-irAE, and unmask a prominent Th1-CD8+ T cell axis in both blood and inflamed joints. CX3CR1hi CD8+ T cells in blood and CXCR3hi CD8+ T cells in synovial fluid, the most clonally expanded T cells, significantly share TCR repertoires. The migration of blood CX3CR1hi CD8+ T cells into joints is possibly mediated by CXCL9/10/11/16 expressed by myeloid cells. Furthermore, arthritis after combined CTLA-4 and PD-1 inhibitor therapy preferentially has enhanced Th17 and transient Th1/Th17 cell signatures. Our data provide insights into the mechanisms, predictive biomarkers, and therapeutic targets for arthritis-irAE.

USP1-trapping lesions as a source of DNA replication stress and genomic instability

The deubiquitinase USP1 is a critical regulator of genome integrity through the deubiquitylation of Fanconi Anemia proteins and the DNA replication processivity factor, proliferating cell nuclear antigen (PCNA). Uniquely, following UV irradiation, USP1 self-inactivates through autocleavage, which enables its own degradation and in turn, upregulates PCNA monoubiquitylation. However, the functional role for this autocleavage event during physiological conditions remains elusive. Herein, we discover that cells harboring an autocleavage-defective USP1 mutant, while still able to robustly deubiquitylate PCNA, experience more replication fork-stalling and premature fork termination events. Using super-resolution microscopy and live-cell single-molecule tracking, we show that these defects are related to the inability of this USP1 mutant to be properly recycled from sites of active DNA synthesis, resulting in replication-associated lesions. Furthermore, we find that the removal of USP1 molecules from DNA is facilitated by the DNA-dependent metalloprotease Spartan to counteract the cytotoxicity caused by “USP1-trapping”. We propose a utility of USP1 inhibitors in cancer therapy based on their ability to induce USP1-trapping lesions and consequent replication stress and genomic instability in cancer cells, similar to how non-covalent DNA-protein crosslinks cause cytotoxicity by imposing steric hindrances upon proteins involved in DNA transactions.

Discovery of Novel Epoxyketone Peptides as Lipase Inhibitors.

Obesity is the most common nutritional disorder in the developed world and is associated with important comorbidities. Pancreatic lipase (PL) inhibitors play a key role in the metabolism of human fat. A series of novel epoxyketones peptide derivatives were investigated for their pancreatic lipase inhibitory activity. The epoxyketone moiety is a well-known reactive electrophile group that has been used as part of proteasome inhibitors in cancer therapy, and it is widely believed that these are very selective for targeting the proteasome active site. Here we investigated various peptide derivatives with an epoxide warhead for their anti-lipase activity. The assessment of these novel epoxyketones was performed by an in-house method that we developed for rapid screening and identification of lipase inhibitors using GC-FID. Herein, we present a novel anti-lipase pharmacophore based on epoxyketone peptide derivatives that showed potent anti-lipase activity. Many of these derivatives had comparable or more potent activity than the clinically used lipase inhibitors such as orlistat. In addition, the lipase appears to be inhibited by a wide range of epoxyketone analogues regardless of the configuration of the epoxide in the epoxyketone moiety. The presented data in this study shows the first example of the use of epoxyketone peptides as novel lipase inhibitors.

A review on cullin neddylation and strategies to identify its inhibitors for cancer therapy.

The cullin-RING E3 ligases (CRLs) are the biggest components of the E3 ubiquitin ligase protein family, and they represent an essential role in various diseases that occur because of abnormal activation, particularly in tumors development. Regulation of CRLs needs neddylation, a post-translational modification involving an enzymatic cascade that transfers small, ubiquitin-like NEDD8 protein to CRLs. Many previous studies have confirmed neddylation as an enticing target for anticancer drug discoveries, and few recent studies have also found a significant increase in advancement in protein neddylation, including preclinical and clinical target validation to discover the neddylation inhibitor compound. In the present review, we first presented briefly the essence of CRLs' neddylation and its control, systematic analysis of CRLs, followed by the description of a few recorded chemical inhibitors of CRLs neddylation enzymes with recent examples of preclinical and clinical targets. We have also listed various structure-based pointing of protein-protein dealings in the CRLs' neddylation reaction, and last, the methods available to discover new inhibitors of neddylation are elaborated. This review will offer a concentrated, up-to-date, and detailed description of the discovery of neddylation inhibitors.

OVERVIEW OF MITOXANTRONE-A POTENTIAL CANDIDATE FOR TREATMENT OF BREAST CANCER

Anthraquinones are one of the popular classes of aromatic compounds which possess potential anticancer properties by suppressing the nucleic acid formation and proteins essential to the survival of cancerous cells. Mitoxantrone (MT) is an antibiotic and antineoplastic agent belonging to the anthracycline class of compounds which exhibit minimal incident of drug resistance. It is a synthetic anticancer drug, bound to enzyme topoisomerase IIα inhibitor, and intercalates DNA topoisomerase IIα, preventing re-ligations in DNA strands fragmentation and disruption of DNA repair. The expression of this enzyme was used tumor cells marker because of its key function in cell proliferation. The cleavable complex of topoisomerase IIα is hypothesized to damage the DNA and may enhance apoptosis in tumor cell proliferation. The susceptibility of cells to mitoxantrone is associated with cell topoisomerase II α protein and lowered resistance in breast cancer line cell lines to topoisomerase IIα inhibitors. MT is an ABC-transporter in breast cancer, also designated to be associated with “Breast cancer resistance protein” (BCRP) and it is also a cell cycle non-specific anti-cancer drug and P-glycoprotein substrate. Multiple drug resistance is one of the major drawbacks of this drug which can be avoided by reducing the efflux of the drug from cancer cells by formulating drug by using lipophilic carriers. This manuscript discusses about MT's source, chemistry, physicochemical properties, anti-cancer effects of mitoxantrone and possible pathways, Mitoxantrone targeting topoisomerase II inhibitor for cancer therapy and its mechanism, Various Nano formulation development strategy, toxicity profile, and a few patents related information.

Protein Tyrosine Phosphatase 1B (PTP1B): Insights into its New Implications in Tumorigenesis.

In vivo, tyrosine phosphorylation is a reversible and dynamic process governed by the opposing activities of protein tyrosine kinases and phosphatases. Defective or inappropriate operation of these proteins leads to aberrant tyrosine phosphorylation, which contributes to the development of many human diseases, including cancers. PTP1B, a non-transmembrane phosphatase, is generally considered a negative regulator of the metabolic signaling pathways and a promising drug target for type II diabetes and obesity. Recently, PTP1B is gaining considerable interest due to its important function and therapeutic potential in other diseases. An increasing number of studies have indicated that PTP1B plays a vital role in the initiation and progression of cancers and could be a target for new cancer therapies. Following recent advances in the aspects mentioned above, this review is focused on the major functions of PTP1B in different types of cancer and the underlying mechanisms behind these functions, as well as the potential pharmacological effects of PTP1B inhibitors in cancer therapy.

Dysregulated lipid metabolism blunts the sensitivity of cancer cells to EZH2 inhibitor.

SummaryBackgroundSensitivity has been a key issue for Enhancer of zeste homolog 2 (EZH2) inhibitors in cancer therapy. The EZH2 inhibitor EPZ-6438 was first approved by the US Food and Drug Administration (FDA) in 2020. However, its inadequate anti-cancer activity in solid tumors limits its clinical application. In this study, we utilized the multiple cancer cell lines, which are less sensitive to the EZH2 inhibitor GSK126, combining animal model and clinical data to investigate the underlying mechanism.MethodsIncuCyte S3 was used to explore the difference in the responsiveness of hematological tumor cells and solid tumor cells to GSK126. Transcriptome and metabolome of B16F10 cells after GSK126 treatment were analyzed and the distinct changes in the metabolic profile were revealed. Real-time quantitative PCR and western blot experiments were used to further verify the multi-omics data. ChIP-qPCR was performed to detected H3K27me3 enrichment of target genes. Finally, the anti-tumor effects of combining GSK126 and lipid metabolism drugs were observed with IncuCyte S3 platform, CCK-8 and animal model respectively.FindingsWe found that although the proliferative phenotype did not show strong difference upon treatment with GSK126, the transcriptome and metabolome changed profoundly. GSK126 treatment led to broad shifts in glucose, amino acid, and lipid metabolism. Lipid synthesis was strengthened manifested by the increasing abundance of unsaturated fatty acids. SCD1 and ELOVL2 were regulated by H3K27me3 at gene regulatory region, and upregulated by EZH2 knockdown and inhibitors. SCD1 knockdown increased cellular sensitivity to GSK126. Based on the findings above, the application of the combination with SCD1 inhibitor significantly attenuated the proliferation of cancer and increased the sensitivity to GSK126 by suppressing desaturation of fatty acids.InterpretationDysregulated lipid metabolism can blunt the sensitivity of cancer cells to GSK126. These characteristics shed light on the novel combination therapy strategies to combat tumor resistance.FundingNational Natural Science Foundation of China (No. 81672091, No.91749107 and No. 81972966).

AKT Isoforms in the Immune Response in Cancer.

AKT is a protein kinasethat exists in three isoforms: AKT1, AKT2, and AKT3. Though similar in structure, these isoforms display different effects. AKT is activated downstream of PI3K, and together, this signaling pathway helps regulate cellular processes including cell growth, proliferation, metabolism, survival, and apoptosis. Disruption in these pathways has been associated with disorders including cardiovascular diseases, developmental disorders, inflammatory responses, autoimmune diseases, neurologic disorders, type 2 diabetes, and several cancers. In cancer, deregulation in the PI3K/AKT pathway can be manifested as tumorigenesis, pathological angiogenesis, and metastasis. Increased activity has been correlated with tumor progression and resistance to cancer treatments. Recent studies have suggested that inhibition of the PI3K/AKT pathway plays a significant role in the development, expansion, and proliferation of cells of the immune system. Additionally, AKT has been found to play an important role in differentiating regulatory T cells, activating B cells, and augmenting tumor immunosurveillance. This emphasizes AKT as a potential target for inhibition in cancer therapy. This chapter reviews AKT structure and regulation, its different isoforms, its role in immune cells, and its modulation in oncotherapy.

DNA-PKcs: A Targetable Protumorigenic Protein Kinase.

DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is a pleiotropic protein kinase that plays critical roles in cellular processes fundamental to cancer. DNA-PKcs expression and activity are frequently deregulated in multiple hematologic and solid tumors and have been tightly linked to poor outcome. Given the potentially influential role of DNA-PKcs in cancer development and progression, therapeutic targeting of this kinase is being tested in preclinical and clinical settings. This review summarizes the latest advances in the field, providing a comprehensive discussion of DNA-PKcs functions in cancer and an update on the clinical assessment of DNA-PK inhibitors in cancer therapy.

20P Identifying novel biomarkers with deep proteomics profiling and survival analysis in NSCLC patients treated with anti-PD-1-blockade

Use of immune checkpoint inhibitors in cancer therapy has significantly improved response and survival rates of patients with various cancer types including NSCLC. However, only a fraction of patients experienced clinical benefit. Existing predictive biomarkers are suboptimal especially when outcomes such as progression free survival (PFS) or overall survival are applied. Hence, the use of novel tools in precision medicine such as state-of-the-art proteomics using data independent acquisition mass spectrometry (DIA-MS) could address some of these drawbacks.

Targeting the DNA damage response: PARP inhibitors and new perspectives in the landscape of cancer treatment.

Cancer derives from alterations of pathways responsible for cell survival, differentiation and proliferation. Dysfunctions of mechanisms protecting genome integrity can promote oncogenesis but can also be exploited as therapeutic target. Poly-ADP-Ribose-Polymerase (PARP)-inhibitors, the first approved targeted agents able to tackle DNA damage response (DDR), have demonstrated antitumor activity, particularly when homologous recombination impairment is present. Despite the relevant results achieved, a large proportion of patients fail to obtain durable responses. The development of innovative treatments, able to overcome resistance and ensure long-lasting benefit for a wider population is still an unmet need. Moreover, improvement in biomarker assays is necessary to properly identify patients who can benefit from DDR targeting agents. Here we summarize the main DDR pathways, explain the current role of PARP inhibitors in cancer therapy and illustrate new therapeutic strategies targeting the DDR, focusing on the combinations of PARP inhibitors with other agents and on cell-cycle checkpoint inhibitors.

Abstract P106: Reprogramming regulatory T cells (Treg) using a MALT1 inhibitor for cancer therapy

Abstract Background Despite transforming effects of immune checkpoint blockade (ICB) therapy, objective response rates are low for most solid tumors. In the tumor microenvironment (TME), regulatory T cells are functionally unstable, likely due to changes in Treg metabolism seen in the tumor milieu. Destabilized Treg are susceptible to reprogramming wherein they can be induced to lose their immunosuppressive function and to secrete interferon-gamma (IFN-g). Thus, Treg reprogramming offers a novel strategy to sensitize unresponsive tumors to ICB. Notably, blockade of MALT1 protease induces Treg reprogramming in the TME but without affecting Treg in healthy tissue. MPT-0118, an orally dosed MALT1 inhibitor, was developed to reprogram Treg in the TME and is currently being assessed in patients with advanced tumors. Approach Treatments included MPT-0118 and anti-PD-1. In vivo studies in mice assessed anti-tumor effects using D4M.3A, B16F10, and MC38 syngeneic tumors. Human and mouse tumor tissues were evaluated for Treg reprogramming by in situ hybridization or flow cytometry. Studies in rats and dogs assessed pharmacokinetics and safety. Results MPT-0118 demonstrated dose-dependent in vivo anti-tumor activity. Consistent with the hypothesis that Treg reprogramming supports anti-tumor immunity by initiating IFN-g-driven tumor inflammation, the effect was strongest in combination with anti-PD-1 and in models that are not responsive to ICB alone. MPT-0118-treated tumors showed an increase in IFN-g-secreting Treg, associated with decelerated tumor growth. Ex vivo, MPT-0118 induced Treg reprogramming in tumors resected from patients with colorectal and endometrial cancers. While MPT-0118 caused Treg to produce IFN-g, no changes in the frequencies of Treg circulating in blood were detected in rats. Modeling of the human effective dose and toxicology studies demonstrate a >2x therapeutic window in patients. Conclusions MPT-0118 Treg reprogramming represents a novel strategy with the potential to improve responses to ICB therapy in solid tumors. A Phase 1/1b dose-escalation and cohort-expansion clinical trial evaluating MPT-0018 is underway. Citation Format: Peter Keller, Irina Mazo, Yun Gao, Vijayapal Reddy, Francisco Caballero, Sam Kazer, Dannah Miller, Roberto Gianani, James E. Marvin, Bret Stephens, Gregory L. Beatty, Ulrich H. von Andrian, Thorsten R. Mempel. Reprogramming regulatory T cells (Treg) using a MALT1 inhibitor for cancer therapy [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P106.

Advances in glioma-associated oncogene (GLI) inhibitors for cancer therapy.

The Hedgehog/Glioma-associated oncogene homolog (HH/GLI) signaling pathway regulates self-renewal of rare and highly malignant cancer stem cells, which have been shown to account for the initiation and maintenance of tumor growth as well as for drug resistance, metastatic spread and relapse. As an important component of the Hh signaling pathway, glioma-associated oncogene (GLI) acts as a key signal transmission hub for various signaling pathways in many tumors. Here, we review direct and indirect inhibitors of GLI; summarize the abundant active structurally diverse natural GLI inhibitors; and discuss how to better develop and utilize GLI inhibitors to solve the problem of drug resistance in tumors of interest. In summary, GLI inhibitors will be promising candidates for various cancer treatments.

Angiogenesis Pathway in Kidney Renal Clear Cell Carcinoma and Its Prognostic Value for Cancer Risk Prediction.

Angiogenesis, a process highly regulated by pro-angiogenic and anti-angiogenic factors, is disrupted and dysregulated in cancer. Despite the increased clinical use of angiogenesis inhibitors in cancer therapy, most molecularly targeted drugs have been less effective than expected. Therefore, an in-depth exploration of the angiogenesis pathway is warranted. In this study, the expression of angiogenesis-related genes in various cancers was explored using The Cancer Genome Atlas datasets, whereupon it was found that most of them were protective genes in the patients with kidney renal clear cell carcinoma (KIRC). We divided the samples from the KIRC dataset into three clusters according to the mRNA expression levels of these genes, with the enrichment scores being in the order of Cluster 2 (upregulated expression) > Cluster 3 (normal expression) > Cluster 1 (downregulated expression). The survival curves plotted for the three clusters revealed that the patients in Cluster 2 had the highest overall survival rates. Via a sensitivity analysis of the drugs listed on the Genomics of Drug Sensitivity in Cancer database, we generated IC50 estimates for 12 commonly used molecularly targeted drugs for KIRC in the three clusters, which can provide a more personalized treatment plan for the patients according to angiogenesis-related gene expression. Subsequently, we investigated the correlation between the angiogenesis pathway and classical cancer-related genes as well as that between the angiogenesis score and immune cell infiltration. Finally, we used the least absolute shrinkage and selection operator (LASSO)–Cox regression analysis to construct a risk score model for predicting the survival of patients with KIRC. According to the areas under the receiver operating characteristic (ROC) curves, this new survival model based on the angiogenesis-related genes had high prognostic prediction value. Our results should provide new avenues for the clinical diagnosis and treatment of patients with KIRC.

Long-Acting Tumor-Activated Prodrug of a TGFβR Inhibitor.

Inhibition of TGFβ signaling in concert with a checkpoint blockade has been shown to provide improved and durable antitumor immune response in mouse models. However, on-target adverse cardiovascular effects have limited the clinical use of TGFβ receptor (TGFβR) inhibitors in cancer therapy. To restrict the activity of TGFβR inhibitors to tumor tissues and thereby widen the therapeutic index, a series of tumor-activated prodrugs of a selective small molecule TGFβR1 inhibitor 1 were prepared by appending 1 to a serine protease substrate and a half-life extension fatty acid carbon chain. The prodrugs were shown to be selectively metabolized in tumor tissues relative to the heart and blood and demonstrated a prolonged favorable increase in the tumor-to-heart ratio of the active drug in tissue distribution studies. Once-weekly administration of the most tissue-selective compound 10 provided anti-tumor efficacy comparable to the parent compound and reduced systemic exposure of the active drug.

Diverse landscape of dermatologic toxicities from small-molecule inhibitor cancer therapy.

Advances in molecular biology and genetics have contributed to breakthrough treatments directed at specific pathways associated with the development of cancer. Small-molecule inhibitors (Nibs) aimed at a variety of cellular pathways have been efficacious; however, they are associated with significant dermatologic toxicities. We conducted a comprehensive review of dermatologic toxicities associated with Nibs categorized into the following five groups: (a) mitogen-activated protein kinase; (b) growth factor/multi-tyrosine kinase; (c) cell division/DNA repair; (d) signaling associated with myeloproliferative neoplasms; and (e) other signaling pathways. Prospective phase I, II, or III clinical trials, retrospective literature reviews, systematic reviews/meta-analyses, and case reviews/reports were included for analysis. Dermatologic toxicities reviewed were associated with every class of Nibs and ranged from mild to severe or life-threatening adverse skin reactions. Inflammatory reactions manifesting as maculopapular, papulopustular/acneiform, and eczematous lesions were frequent types of dermatologic toxicities seen with Nibs. Squamous cell carcinoma with keratoacanthoma-like features was associated with a subset of Nibs. Substantial overlap in dermatologic toxicities was found between Nibs. Dermatologic toxicities from Nibs are diverse and may overlap between classes of Nibs. Recognition of the various types of toxicities from Nibs is critical for patient care in the era of "oncodermatology/dermatopathology."

Cytosolic delivery of CDK4/6 inhibitor p16 protein using engineered protein crystals for cancer therapy

The tumor suppressor p16 protein is an endogenous CDK4/6 inhibitor. Inactivation of its encoding gene is found in nearly half of human cancers. Restoration of p16 function via adenovirus-based gene delivery has been shown to be effective in suppressing aberrant cell growth in many types of cancer, however, the potential risk of insertional mutagenesis in genomic DNA remains a major concern. Thus, there has been great interest in developing efficient strategies to directly deliver proteins into cells as an alternative that can avoid such safety concerns while achieving a comparable therapeutic effect. Nevertheless, intracellular delivery of protein therapeutics remains a challenge. Our group has recently developed a protein delivery platform based on an engineered Pos3Aa protein that forms sub-micrometer-sized crystals in Bacillus thuringiensis cells. In this report, we describe the further development of this platform (Pos3AaTM) via rationally designed site-directed mutagenesis, and its resultant potency for the delivery of cargo proteins into cells. Pos3AaTM-based fusion protein crystals are shown to exhibit improved release of their cargo proteins as demonstrated using a model mCherry protein. Importantly, this Pos3AaTM platform is able to mediate the efficient intracellular delivery of p16 protein with significant endosomal escape, resulting in p16-mediated inhibition of CDK4/6 kinase activity and Rb phosphorylation, and as a consequence, significant cell cycle arrest and cell growth inhibition. These results validate the ability of these improved Pos3AaTM crystals to mediate enhanced cytosolic protein delivery and highlight the potential of using protein therapeutics as selective CDK4/6 inhibitors for cancer therapy. Statement of significanceCytosolic delivery of bioactive therapeutic proteins capable of eliciting therapeutic benefit remains a significant challenge. We have previously developed a protein delivery platform based on engineered Pos3Aa protein crystals with excellent cell-permeability and endosomal escape properties. In this report, we describe the rational design of an improved Pos3Aa triple mutant (Pos3AaTM) with enhanced cargo release. We demonstrate that Pos3AaTM-mCherry-p16 fusion crystals can efficiently deliver p16 protein, a CDK4/6 inhibitor frequently inactivated in human cancers, into p16-deficient UM-SCC-22A cells, where it promotes significant G1 cell cycle arrest and cell growth inhibition. These results highlight the ability of the Pos3AaTM platform to promote potent cytosolic delivery of protein therapeutics, and the efficacy of p16 protein delivery as an effective strategy for treating cancer.