HTS discovery of PARP1-HPF1 complex inhibitors in cancer.

PARP1/2 inhibitors (PARPi) are effective clinically used drugs for the treatment of cancers with BRCA deficiencies. PARPi have had limited success and applicability beyond BRCA deficient cancers, and their effect is diminished by resistance mechanisms. The recent discovery of Histone PARylation Factor (HPF1) and the role it plays in the PARylation reaction by forming a shared active site with PARP1 raises the possibility that novel inhibitors that target the PARP1-HPF1 complex can be identified. Herein we describe a simple and cost-effective high-throughput screening (HTS) method aimed at discovering inhibitors of the PARP1-HPF1 complex. Upon HTS validation, we first applied this method to screen a small PARP-focused library of compounds and then scale up our approach using robotic automation to conduct a pilot screen of 10,000 compounds and validating >100 hits. This work demonstrates for the first time the capacity to discover potent inhibitors of the PARP1-HPF1 complex, which may have utility as probes to better understand the DNA damage response and as therapeutics for cancer.

Surfaceome Profiling of Cell Lines and Patient-Derived Xenografts Confirm FGFR4, NCAM1, CD276, and Highlight AGRL2, JAM3, and L1CAM as Surface Targets for Rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. The prognosis for patients with high-grade and metastatic disease is still very poor, and survivors are burdened with long-lasting side effects. Therefore, more effective and less toxic therapies are needed. Surface proteins are ideal targets for antibody-based therapies, like bispecific antibodies, antibody-drug conjugates, or chimeric antigen receptor (CAR) T-cells. Specific surface targets for RMS are scarce. Here, we performed a surfaceome profiling based on differential centrifugation enrichment of surface/membrane proteins and detection by LC-MS on six fusion-positive (FP) RMS cell lines, five fusion-negative (FN) RMS cell lines, and three RMS patient-derived xenografts (PDXs). A total of 699 proteins were detected in the three RMS groups. Ranking based on expression levels and comparison to expression in normal MRC-5 fibroblasts and myoblasts, followed by statistical analysis, highlighted known RMS targets such as FGFR4, NCAM1, and CD276/B7-H3, and revealed AGRL2, JAM3, MEGF10, GPC4, CADM2, as potential targets for immunotherapies of RMS. L1CAM expression was investigated in RMS tissues, and strong L1CAM expression was observed in more than 80% of alveolar RMS tumors, making it a practicable target for antibody-based therapies of alveolar RMS.

GSDMD-dependent pyroptotic induction by a multivalent CXCR4-targeted nanotoxin blocks colorectal cancer metastases.

Colorectal cancer (CRC) remains the third cause of cancer-related mortality in Western countries, metastases are the main cause of death. CRC treatment remains limited by systemic toxicity and chemotherapy resistance. Therefore, nanoparticle-mediated delivery of cytotoxic agents selectively to cancer cells represents an efficient strategy to increase the therapeutic index and overcome drug resistance. We have developed the T22-PE24-H6 therapeutic protein-only nanoparticle that incorporates the exotoxin A from Pseudomonas aeruginosa to selectively target CRC cells because of its multivalent ligand display that triggers a high selectivity interaction with the CXCR4 receptor overexpressed on the surface of CRC stem cells. We here observed a CXCR4-dependent cytotoxic effect for T22-PE24-H6, which was not mediated by apoptosis, but instead capable of inducing a time-dependent and sequential activation of pyroptotic markers in CRC cells in vitro. Next, we demonstrated that repeated doses of T22-PE24-H6 inhibit tumor growth in a subcutaneous CXCR4+ CRC model, also through pyroptotic activation. Most importantly, this nanoparticle also blocked the development of lymphatic and hematogenous metastases, in a highly aggressive CXCR4+ SW1417 orthotopic CRC model, in the absence of systemic toxicity. This targeted drug delivery approach supports for the first time the clinical relevance of inducing GSDMD-dependent pyroptosis, a cell death mechanism alternative to apoptosis, in CRC models, leading to the selective elimination of CXCR4+ cancer stem cells, which are associated with resistance, metastases and anti-apoptotic upregulation.

A refined cocktailing of pro-apoptotic nanoparticles boosts anti-tumor activity.

A functional 29 amino acid-segment of the helix α5 from the human BAX protein has been engineered for production in recombinant bacteria as self-assembling, GFP-containing fluorescent nanoparticles, which are targeted to the tumoral marker CXCR4. These nanoparticles, of around 34 nm in diameter, show a moderate tumor biodistribution and limited antitumoral effect when systemically administered to mouse models of human CXCR4+ colorectal cancer (at 300 µg dose). However, if such BAX nanoparticles are co-administered in cocktail with equivalent nanoparticulate versions of BAK and PUMA proteins at the same total protein dose (300 µg), protein biodistribution and stability in tumor is largely improved, as determined by fluorescence profiles. This fact leads to a potent and faster destruction of tumor tissues when compared to individual pro-apoptotic factors. The analysis and interpretation of the boosted effect, from both the structural and functional sides, offers clues for the design of more efficient nanomedicines and theragnostic agents in oncology based on precise cocktails of human proteins. STATEMENT OF SIGNIFICANCE: Several human pro-apoptotic peptides (namely BAK, BAX and PUMA) have been engineered as self-assembling protein nanoparticles targeted to the tumoral marker CXCR4. The systemic administration of the same final amounts of those materials as single drugs, or as combinations of two or three of them, shows disparate intensities of antitumoral effects in a mouse model of human colorectal cancer, which are boosted in the triple combination on a non-additive basis. The superiority of the combined administration of pro-apoptotic agents, acting at different levels of the apoptotic cascade, opens a plethora of possibilities for the development of effective and selective cancer therapies based on the precise cocktailing of pro-apoptotic nanoparticulate agents.

Engineering Secretory Amyloids for Remote and Highly Selective Destruction of Metastatic Foci.

Functional amyloids produced in bacteria as nanoscale inclusion bodies are intriguing but poorly explored protein materials with wide therapeutic potential. Since they release functional polypeptides under physiological conditions, these materials can be potentially tailored as mimetic of secretory granules for slow systemic delivery of smart protein drugs. To explore this possibility, bacterial inclusion bodies formed by a self-assembled, tumor-targeted Pseudomonas exotoxin (PE24) are administered subcutaneously in mouse models of human metastatic colorectal cancer, for sustained secretion of tumor-targeted therapeutic nanoparticles. These proteins are functionalized with a peptidic ligand of CXCR4, a chemokine receptor overexpressed in metastatic cancer stem cells that confers high selective cytotoxicity in vitro and in vivo. In the mouse models of human colorectal cancer, time-deferred anticancer activity is detected after the subcutaneous deposition of 500 µg of PE24-based amyloids, which promotes a dramatic arrest of tumor growth in the absence of side toxicity. In addition, long-term prevention of lymphatic, hematogenous, and peritoneal metastases is achieved. These results reveal the biomedical potential and versatility of bacterial inclusion bodies as novel tunable secretory materials usable in delivery, and they also instruct how therapeutic proteins, even with high functional and structural complexity, can be packaged in this convenient format.

Collaborative membrane activity and receptor-dependent tumor cell targeting for precise nanoparticle delivery in CXCR4+ colorectal cancer.

By the appropriate selection of functional peptides and proper accommodation sites, we have generated a set of multifunctional proteins that combine selectivity for CXCR4+ cell binding and relevant endosomal escape capabilities linked to the viral peptide HA2. In particular, the construct T22-GFP-HA2-H6 forms nanoparticles that upon administration in mouse models of human, CXCR4+ colorectal cancer, accumulates in primary tumor at levels significantly higher than the parental T22-GFP-H6 HA2-lacking version. The in vivo application of a CXCR4 antagonist has confirmed the prevalence of the CXCR4+ tumor tissue selectivity over unspecific cell penetration, upon systemic administration of the material. Such specificity is combined with improved endosomal escape, what overall results in a precise and highly efficient tumor biodistribution. These data strongly support the functional recruitment as a convenient approach to generate protein materials for clinical applications. More precisely, they also support the unexpected concept that enhancing the unspecific membrane activity of a protein material does not necessarily compromise, but it can even improve, the selective cell targeting offered by an accompanying functional module. STATEMENT OF SIGNIFICANCE: We have shown here that the combination of cell-penetrating and tumor cell-targeting peptides dramatically enhances precise tumor accumulation of protein-only nanoparticles intended for selective drug delivery, in mouse models of human colorectal cancer. This fact is a step forward for the rational design of multifunctional protein nanomaterials for improved cancer therapies.

Selective depletion of metastatic stem cells as therapy for human colorectal cancer.

Selective elimination of metastatic stem cells (MetSCs) promises to block metastatic dissemination. Colorectal cancer (CRC) cells overexpressing CXCR4 display trafficking functions and metastasis-initiating capacity. We assessed the antimetastatic activity of a nanoconjugate (T22-GFP-H6-FdU) that selectively delivers Floxuridine to CXCR4+ cells. In contrast to free oligo-FdU, intravenous T22-GFP-H6-FdU selectively accumulates and internalizes in CXCR4+ cancer cells, triggering DNA damage and apoptosis, which leads to their selective elimination and to reduced tumor re-initiation capacity. Repeated T22-GFP-H6-FdU administration in cell line and patient-derived CRC models blocks intravasation and completely prevents metastases development in 38-83% of mice, while showing CXCR4 expression-dependent and site-dependent reduction in foci number and size in liver, peritoneal, or lung metastases in the rest of mice, compared to free oligo-FdU. T22-GFP-H6-FdU induces also higher regression of established metastases than free oligo-FdU, with negligible distribution or toxicity in normal tissues. This targeted drug delivery approach yields potent antimetastatic effect, through selective depletion of metastatic CXCR4+ cancer cells, and validates metastatic stem cells (MetSCs) as targets for clinical therapy.

Release of targeted protein nanoparticles from functional bacterial amyloids: A death star-like approach.

Sustained release of drug delivery systems (DDS) has the capacity to increase cancer treatment efficiency in terms of drug dosage reduction and subsequent decrease of deleterious side effects. In this regard, many biomaterials are being investigated but none offers morphometric and functional plasticity and versatility comparable to protein-based nanoparticles (pNPs). Here we describe a new DDS by which pNPs are fabricated as bacterial inclusion bodies (IB), that can be easily isolated, subcutaneously injected and used as reservoirs for the sustained release of targeted pNPs. Our approach combines the high performance of pNP, regarding specific cell targeting and biodistribution with the IB supramolecular organization, stability and cost effectiveness. This renders a platform able to provide a sustained source of CXCR4-targeted pNPs that selectively accumulate in tumor cells in a CXCR4+ colorectal cancer xenograft model. In addition, the proposed system could be potentially adapted to any other protein construct offering a plethora of possible new therapeutic applications in nanomedicine.

Self-assembling toxin-based nanoparticles as self-delivered antitumoral drugs.

Loading capacity and drug leakage from vehicles during circulation in blood is a major concern when developing nanoparticle-based cell-targeted cytotoxics. To circumvent this potential issue it would be convenient the engineering of drugs as self-delivered nanoscale entities, devoid of any heterologous carriers. In this context, we have here engineered potent protein toxins, namely segments of the diphtheria toxin and the Pseudomonas aeruginosa exotoxin as self-assembling, self-delivered therapeutic materials targeted to CXCR4+ cancer stem cells. The systemic administration of both nanostructured drugs in a colorectal cancer xenograft mouse model promotes efficient and specific local destruction of target tumor tissues and a significant reduction of the tumor volume. This observation strongly supports the concept of intrinsically functional protein nanoparticles, which having a dual role as drug and carrier, are designed to be administered without the assistance of heterologous vehicles.

Cancer-specific uptake of a liganded protein nanocarrier targeting aggressive CXCR4+ colorectal cancer models.

Unliganded drug-nanoconjugates accumulate passively in the tumor whereas liganded nanoconjugates promote drug internalization in tumor cells via endocytosis and increase antitumor efficacy. Whether or not tumor cell internalization associates with enhanced tumor uptake is still under debate. We here compared tumor uptake of T22-GFP-H6, a liganded protein carrier targeting the CXCR4 receptor, and the unliganded GFP-H6 carrier in subcutaneous and metastatic colorectal cancer models. T22-GFP-H6 had a higher tumor uptake in primary tumor and metastatic foci than GFP-H6, with no biodistribution or toxicity on normal tissues. T22-GFP-H6 was detected in target CXCR4+ tumor cell cytosol whereas GFP-H6 was detected in tumor stroma. SDF1-α co-administration switched T22-GFP-H6 internalization from CXCR4+ tumor epithelial cells to the stroma. Therefore, the incorporation of a targeting ligand promotes selective accumulation of the nanocarrier inside target tumor cells while increasing whole tumor uptake in a CXCR4-dependent manner, validating T22-GFP-H6 as a CXCR4-targeted drug carrier.