In Silico Transcriptomic Expression of MSR1 in Solid Tumors Is Associated with Responses to Anti-PD1 and Anti-CTLA4 Therapies.

Immuno-oncology has gained momentum with the approval of antibodies with clinical activities in different indications. Unfortunately, for anti-PD (L)1 agents in monotherapy, only half of the treated population achieves a clinical response. For other agents, such as anti-CTLA4 antibodies, no biomarkers exist, and tolerability can limit administration. In this study, using publicly available genomic datasets, we evaluated the expression of the macrophage scavenger receptor-A (SR-A) (MSR1) and its association with a response to check-point inhibitors (CPI). MSR1 was associated with the presence of macrophages, dendritic cells (DCs) and neutrophils in most of the studied indications. The presence of MSR1 was associated with macrophages with a pro-tumoral phenotype and correlated with TIM3 expression. MSR1 predicted favorable overall survival in patients treated with anti-PD1 (HR: 0.56, FDR: 1%, p = 2.6 × 10-5), anti PD-L1 (HR: 0.66, FDR: 20%, p = 0.00098) and anti-CTLA4 (HR: 0.37, FDR: 1%, p = 4.8 × 10-5). When specifically studying skin cutaneous melanoma (SKCM), we observed similar effects for anti-PD1 (HR: 0.65, FDR: 50%, p = 0.0072) and anti-CTLA4 (HR: 0.35, FDR: 1%, p = 4.1 × 10-5). In a different dataset of SKCM patients, the expression of MSR1 predicted a clinical response to anti-CTLA4 (AUC: 0.61, p = 2.9 × 10-2). Here, we describe the expression of MSR1 in some solid tumors and its association with innate cells and M2 phenotype macrophages. Of note, the presence of MSR1 predicted a response to CPI and, particularly, anti-CTLA4 therapies in different cohorts of patients. Future studies should prospectively explore the association of MSR1 expression and the response to anti-CTLA4 strategies in solid tumors.

Genomic and Immunologic Correlates in Prostate Cancer with High Expression of KLK2.

The identification of surfaceome proteins is a main goal in cancer research to design antibody-based therapeutic strategies. T cell engagers based on KLK2, a kallikrein specifically expressed in prostate cancer (PRAD), are currently in early clinical development. Using genomic information from different sources, we evaluated the immune microenvironment and genomic profile of prostate tumors with high expression of KLK2. KLK2 was specifically expressed in PRAD but it was not significant associated with Gleason score. Additionally, KLK2 expression did not associate with the presence of any immune cell population and T cell activating markers. A mild correlation between the high expression of KLK2 and the deletion of TMPRSS2 was identified. KLK2 expression associated with high levels of surface proteins linked with a detrimental response to immune checkpoint inhibitors (ICIs) including CHRNA2, FAM174B, OR51E2, TSPAN1, PTPRN2, and the non-surface protein TRPM4. However, no association of these genes with an outcome in PRAD was observed. Finally, the expression of these genes in PRAD did not associate with an outcome in PRAD and any immune populations. We describe the immunologic microenvironment on PRAD tumors with a high expression of KLK2, including a gene signature linked with an inert immune microenvironment, that predicts the response to ICIs in other tumor types. Strategies targeting KLK2 with T cell engagers or antibody-drug conjugates will define whether T cell mobilization or antigen release and stimulation of immune cell death are sufficient effects to induce clinical activity.

Considerations for the design of antibody drug conjugates (ADCs) for clinical development: lessons learned.

Antibody-drug conjugates (ADCs) have emerged as a novel therapeutic strategy that has successfully reached patient treatment in different clinical scenarios. ADCs are formed by an antibody against a specific tumor-associated antigen (TAA), a cytotoxic payload, and a chemical linker that binds both. To this regard, most efforts have been focused on target identification, antibody design and linker optimization, but other relevant aspects for clinical development have not received the necessary attention. In this article using data from approved ADCs, we evaluated all characteristics of these agents, including payload physicochemical properties, in vitro potency, drug antibody ratio (DAR), exposure-response relationships, and clinical development strategies. We suggest that compounds with best options for clinical development include those with optimal payload physicochemical properties and cleavable linkers that would lead to a bystander effect. These modalities can facilitate the development of ADCs in indications with low expression of the TAA. Early clinical development strategies including changes in the schedule of administration with more frequent doses are also discussed in the context of an efficient strategy. In conclusion, we highlight relevant aspects that are needed for the optimal development of ADCs in cancer, proposing options for improvement.

Guanylation Reactions for the Rational Design of Cancer Therapeutic Agents.

The modular synthesis of the guanidine core by guanylation reactions using commercially available ZnEt2 as a catalyst has been exploited as a tool for the rapid development of antitumoral guanidine candidates. Therefore, a series of phenyl-guanidines were straightforwardly obtained in very high yields. From the in vitro assessment of the antitumoral activity of such structurally diverse guanidines, the guanidine termed ACB3 has been identified as the lead compound of the series. Several biological assays, an estimation of AMDE values, and an uptake study using Fluorescence Lifetime Imaging Microscopy were conducted to gain insight into the mechanism of action. Cell death apoptosis, induction of cell cycle arrest, and reduction in cell adhesion and colony formation have been demonstrated for the lead compound in the series. In this work, and as a proof of concept, we discuss the potential of the catalytic guanylation reactions for high-throughput testing and the rational design of guanidine-based cancer therapeutic agents.

Uncovering therapeutic opportunities in the clinical development of antibody-drug conjugates.

INTRODUCTION: Antibody-drug conjugates (ADCs) are a family of therapeutic agents that have demonstrated clinical activity in several indications. MATERIAL AND METHODS: In this article, we performed a deep analysis of their clinical landscape matched with public genomic human datasets from tumour antigen targets (TATs), to identify empty areas for clinical development. RESULTS: We observed that TATs used in haematological malignancies were more specific than the ones developed in solid cancers. Those included CD19, CD22, CD30, CD33 and CD79b. In solid tumours, we identified TATs, with approved ADCs, widely expressed in non-explored niche indications like Enfortumab vedotin (anti-Nectin4) in lung or cervical cancer; Tisotumab vedotin (anti-TF) in glioblastoma or pancreatic cancer; and Sacituzumab govitecan (anti-TROP2) in pancreatic, gastric, thyroid or endometrial cancer, among others. Similarly, niche indications for ADCs in clinical development included targets for CD71, PSMA, PTK7 or CD74, in tumours like breast, lung, stomach or colon. Some of these TATs were essential for the survival of tumour cells like CD71, PSMA and PTK7. CONCLUSIONS: In summary, our study opens the door for further evaluation of ADCs in several indications not explored before.

Genomic Mapping of Epidermal Growth Factor Receptor and Mesenchymal-Epithelial Transition-Up-Regulated Tumors Identifies Novel Therapeutic Opportunities.

BACKGROUND: The identification of proteins in the cellular membrane of the tumoral cell is a key to the design of therapeutic agents. Recently, the bi-specific antibody amivantamab, targeting the oncogenic membrane proteins EGFR and MET, received regulatory approval for the treatment of adult patients with locally advanced or metastatic NSCLC. METHODS: The authors interrogated several publicly available genomic datasets to evaluate the expression of both receptors and PD-L1 in most of the solid and hematologic malignancies and focused on prostate adenocarcinoma (PRAD) and pancreatic adenocarcinoma (PAAD). RESULTS: In PAAD, EGFR highly correlated with PD-L1 and MET, and MET showed a moderate correlation with PD-L1, while in PRAD, EGFR, MET and PD-L1 showed a strong correlation. In addition, in tumors treated with immune checkpoint inhibitors, including anti-PD(L)1 and anti-CTLA4, a high expression of EGFR and MET predicted detrimental survival. When exploring the relationship of immune populations with these receptors, the authors observed that in PAAD and PRAD, EGFR moderately correlated with CD8+ T cells. Furthermore, EGFR and MET correlated with neutrophils in PRAD. CONCLUSIONS: The authors identified tumor types where EGFR and MET were highly expressed and correlated with a high expression of PD-L1, opening the door for the future combination of bi-specific EGFR/MET antibodies with anti-PD(L)1 inhibitors.

Mapping Immune Correlates and Surfaceome Genes in BRAF Mutated Colorectal Cancers.

Despite the impressive results obtained with immunotherapy in several cancer types, a significant fraction of patients remains unresponsive to these treatments. In colorectal cancer (CRC), B-RafV600 mutations have been identified in 8-15% of the patients. In this work we interrogated a public dataset to explore the surfaceome of these tumors and found that several genes, such as GP2, CLDN18, AQP5, TM4SF4, NTSR1, VNN1, and CD109, were upregulated. By performing gene set enrichment analysis, we also identified a striking upregulation of genes (CD74, LAG3, HLA-DQB1, HLA-DRB5, HLA-DMA, HLA-DMB, HLA-DPB1, HLA-DRA, HLA-DOA, FCGR2B, HLA-DQA1, HLA-DRB1, and HLA-DPA1) associated with antigen processing and presentation via MHC class II. Likewise, we found a strong correlation between PD1 and PD(L)1 expression and the presence of genes encoding for proteins involved in antigen presentation such as CD74, HLA-DPA1, and LAG3. Furthermore, a similar association was observed for the presence of dendritic cells and macrophages. Finally, a low but positive relationship was observed between tumor mutational burden and neoantigen load. Our findings support the idea that a therapeutic strategy based on the targeting of PD(L)1 together with other receptors also involved in immuno-modulation, such as LAG3, could help to improve current treatments against BRAF-mutated CRC tumors.

Antitumoral Activity of a CDK9 PROTAC Compound in HER2-Positive Breast Cancer.

Cyclin-dependent kinases (CDKs) are a broad family of proteins involved in the cell cycle and transcriptional regulation. In this article, we explore the antitumoral activity of a novel proteolysis-targeting chimera (PROTAC) compound against CDK9. Breast cancer cell lines from different subtypes were used. Transcriptomic mapping of CDKs in breast cancer demonstrated that the expression of CDK9 predicted a detrimental outcome in basal-like tumors (HR = 1.51, CI = 1.08-2.11, p = 0.015) and, particularly, in the luminal B subtype with HER2+ expression (HR = 1.82, CI = 1.17-2.82, p = 0.0069). The novel CDK9 PROTAC, THAL-SNS-032, displayed a profound inhibitory activity in MCF7, T47D, and BT474 cells, with less effect in SKBR3, HCC1569, HCC1954, MDA-MB-231, HS578T, and BT549 cells. The three cell lines with HER2 overexpression and no presence of ER, SKBR3, HCC1569, and HCC1954 displayed an EC50 three times higher compared to ER-positive and dual ER/HER2-positive cell lines. BT474-derived trastuzumab-resistant cell lines displayed a particular sensitivity to THAL-SNS-032. Western blot analyses showed that THAL-SNS-032 caused a decrease in CDK9 levels in BT474, BT474-RH, and BT474-TDM1R cells, and a significant increase in apoptosis. Experiments in animals demonstrated an inverse therapeutic index of THAL-SNS-032, with doses in the nontherapeutic and toxic range. The identified toxicity was mainly due to an on-target off-tumor effect of the compound in the gastrointestinal epithelium. In summary, the potent and efficient antitumoral properties of the CDK9 PROTAC THAL-SNS-032 opens the possibility of using this type of compound in breast cancer only if specifically delivered to cancer cells, particularly in ER/HER2-positive and HER2-resistant tumors.

Clinical considerations for the design of PROTACs in cancer.

Degradation of targeted proteins using proteolysis targeting chimeras (PROTACs) has gained momentum. A PROTAC is a bifunctional molecule that consists of three parts: a ligand that interacts with the protein to be degraded, another ligand that binds to an E3 ubiquitin ligase and a linker that connects both. Identification of the right proteins as targets to be degraded and a ligase that is highly expressed in tumors compare with normal tissue is mandatory, as can augment efficacy reducing toxicity. In this article we review the current development stage of PROTACs in cancer to categorize the best PROTAC construction. Targets including BCL2, CDK4 and MCL1 were highly expressed in all tumors; MCL1 was significantly increased in breast cancer and lung adenocarcinoma and CDK4 in colon adenocarcinoma. Degradation of CDK9, AURKA or PLK1, followed by BCL2, MCL1, PTPN11, BRD4, PTK2, showed a high dependency. Most ligases evaluated were not highly present in tumors except for MDM2 in breast, lung, prostate and gastric cancer. In non-transformed tissue MDM2 was the most abundant ligase, followed by cIAP and CRBN, and those with low expression included XIAP and VHL. MDM2 ligase coupled with inhibitors of the targets BCL2, BRD4, CDK9, PLK1 and MCL1 in stomach tumor, and MDM2 with PIK3C3 inhibitors in breast cancer, seems to be the best therapeutic strategy. Our results suggest potential options for the design of PROTACS in specific medical indications.

Transcriptomic Profiles of CD47 in Breast Tumors Predict Outcome and Are Associated with Immune Activation.

Targeting the innate immune system has attracted attention with the development of anti- CD47 antibodies. Anti-CD47 antibodies block the inhibition of the phagocytic activity of macrophages caused by the up-regulation of CD47 on tumor cells. In this study, public genomic data was used to identify genes highly expressed in breast tumors with elevated CD47 expression and analyzed the association between the presence of tumor immune infiltrates and the expression of the selected genes. We found that 142 genes positively correlated with CD47, of which 83 predicted favorable and 32 detrimental relapse-free survival (RFS). From those associated with favorable RFS, we selected the genes with immunologic biological functions and defined a CD47-immune signature composed of PTPRC, HLA-E, TGFBR2, PTGER4, ETS1, and OPTN. In the basal-like and HER2+ breast cancer subtypes, the expression of the CD47-immune signature predicted favorable outcome, correlated with the presence of tumor immune infiltrates, and with gene expression signatures of T cell activation. Moreover, CD47 up-regulated genes associated with favorable survival correlated with pro-tumoral macrophages. In summary, we described a CD47-immune gene signature composed of 6 genes associated with favorable prognosis, T cell activation, and pro-tumoral macrophages in breast cancer tumors expressing high levels of CD47.

MZ1 co-operates with trastuzumab in HER2 positive breast cancer.

BACKGROUND: Although the anti-HER2 antibody trastuzumab augments patient survival in HER2+ breast cancer, a relevant number of patients progress to this treatment. In this context, novel drug combinations are needed to increase its antitumor activity. In this work, we have evaluated the efficacy of proteolysis targeting chimera (PROTAC) compounds based on BET inhibitors (BETi) to augment the activity of trastuzumab in HER2+ breast cancer models. METHODS: BT474 and SKBR3 HER2+ breast cancer cell lines were used. The effects of trastuzumab and the BET-PROTAC MZ1 either alone or in combination, were evaluated using MTT proliferation assays, three-dimensional invasion and adhesion cultures, flow cytometry, qPCR and Western blot. In vivo studies were carried out in a xenografted model in mice. Finally, a Clariom_S_Human transcriptomic array was applied to identify deregulated genes after treatments. RESULTS: MZ1 induced a higher antiproliferative effect compared to the BETi JQ1. The combination of MZ1 and -trastuzumab significantly decreased cell proliferation, the formation of three-dimensional structures and cellular invasion compared to either of the drugs alone. Evaluation of apoptosis resulted in an increase of cell death following treatment with the combination, and biochemical studies displayed modifications of apoptosis and DNA damage components. In vivo administration of agents alone or combined, to tumors orthotopically xenografted in mice, resulted in a decrease of the tumor volume only after MZ1-Trastuzumab combination treatment. Results from a transcriptomic array indicated a series of newly described transcription factors including HOXB7, MEIS2, TCERG1, and DNAJC2, that were associated to poor outcome in HER2+ breast cancer subtype and downregulated by the MZ1-trastuzumab combination. CONCLUSIONS: We describe an active novel combination that includes the BET-PROTAC MZ1 and trastuzumab, in HER2+ tumors. Further studies should be performed to confirm these findings and pave the way for their future clinical development.

Checkpoint Kinase 1 Pharmacological Inhibition Synergizes with DNA-Damaging Agents and Overcomes Platinum Resistance in Basal-Like Breast Cancer.

Basal-like breast cancer is an incurable disease with limited therapeutic options, mainly due to the frequent development of anti-cancer drug resistance. Therefore, identification of druggable targets to improve current therapies and overcome these resistances is a major goal. Targeting DNA repair mechanisms has reached the clinical setting and several strategies, like the inhibition of the CHK1 kinase, are currently in clinical development. Here, using a panel of basal-like cancer cell lines, we explored the synergistic interactions of CHK1 inhibitors (rabusertib and SAR020106) with approved therapies in breast cancer and evaluated their potential to overcome resistance. We identified a synergistic action of these inhibitors with agents that produce DNA damage, like platinum compounds, gemcitabine, and the PARP inhibitor olaparib. Our results demonstrated that the combination of rabusertib with these chemotherapies also has a synergistic impact on tumor initiation, invasion capabilities, and apoptosis in vitro. We also revealed a biochemical effect on DNA damage and caspase-dependent apoptosis pathways through the phosphorylation of H2AX, the degradation of full-length PARP, and the increase of caspases 3 and 8 activity. This agent also demonstrated synergistic activity in a platinum-resistant cell line, inducing an increase in cell death in response to cisplatin only when combined with rabusertib, while no toxic effect was found on non-tumorigenic breast tissue-derived cell lines. Lastly, the combination of CHK1 inhibitor with cisplatin and gemcitabine resulted in more activity than single or double combinations, leading to a higher apoptotic effect. In conclusion, in our study we identify therapeutic options for the clinical development of CHK1 inhibitors, and confirm that the inhibition of this kinase can overcome acquired resistance to cisplatin.

Inhibition of the mitotic kinase PLK1 overcomes therapeutic resistance to BET inhibitors in triple negative breast cancer.

The inhibition of bromo- and extraterminal domains (BET) has shown an anti-proliferative effect in triple negative breast cancer (TNBC). In this article we explore mechanisms of resistance to BET inhibitors (BETi) in TNBC, with the aim of identifying novel ways to overcome such resistance. Two cellular models of acquired resistance to the BET inhibitor JQ1 were generated using a pulsed treatment strategy. MTT, colony formation, and cytometry assays revealed that BETi-resistant cells were particularly sensitive to PLK1 inhibition. Targeting of the latter reduced cell proliferation, especially in resistant cultures. Quantitative PCR analysis of a panel of mitotic kinases uncovered an increased expression of AURKA, TTK, and PLK1, confirmed by Western blot. Only pharmacological inhibition of PLK1 showed anti-proliferative activity on resistant cells, provoking G2/M arrest, increasing expression levels of cyclin B, pH3 and phosphorylation of Bcl-2 proteins, changes that were accompanied by induction of caspase-dependent apoptosis. JQ1-resistant cells orthotopically xenografted into the mammary fat pad of mice led to tumours that retained JQ1-resistance. Administration of the PLK1 inhibitor volasertib resulted in tumour regression. These findings open avenues to explore the future use of PLK1 inhibitors in the clinical setting of BETi-resistant patients.

Identification of a stemness-related gene panel associated with BET inhibition in triple negative breast cancer.

PURPOSE: Triple negative breast cancers (TNBCs) are enriched in cells bearing stem-like features, i.e., cancer stem cells (CSCs), which underlie cancer progression. Thus, targeting stemness may be an interesting treatment approach. The epigenetic machinery is crucial for maintaining the stemness phenotype. Bromodomain and extra-terminal domain (BET) epigenetic reader family members are emerging as novel targets for cancer therapy, and have already shown preclinical effects in breast cancer. Here, we aimed to evaluate the effect of the BET inhibitor JQ1 on stemness in TNBC. METHODS: Transcriptomic, functional annotation and qRT-PCR studies were performed on JQ1-exposed TNBC cells in culture. The results obtained were confirmed in spheroids and spheroid-derived tumours. In addition, limiting dilution, secondary and tertiary tumour sphere formation, matrigel invasion, immunofluorescence and flow cytometry assays were performed to evaluate the effect of JQ1 on CSC features. For clinical outcome analyses, the online tool Kaplan-Meier Plotter and an integrated response database were used. RESULTS: We found that JQ1 modified the expression of stemness-related genes in two TNBC-derived cell lines, MDA-MB-231 and BT549. Among these changes, the CD44 Antigen/CD24 Antigen (CD44/CD24) ratio and Aldehyde Dehydrogenase 1 Family Member A1 (ALDH1A1) expression level, i.e., both classical stemness markers, were found to be decreased by JQ1. Using a validated spheroid model to mimic the intrinsic characteristics of CSCs, we found that JQ1 decreased surface CD44 expression, inhibited self-renewal and invasion, and induced cell cycle arrest in G0/G1, thereby altering the stemness phenotype. We also found associations between four of the identified stemness genes, Gap Junction Protein Alpha 1 (GJA1), CD24, Epithelial Adhesion Molecule (EPCAM) and SRY-related HMG-box gene 9 (SOX9), and a worse TNBC patient outcome. The expression of another two of the stemness-related genes was found to be decreased by JQ1, i.e., ATP Binding Cassette Subfamily G Member 2 (ABCG2) and RUNX2, and predicted a low response to chemotherapy in TNBC patients, which supports a role for RUNX2 as a potential predictive marker for chemotherapy response in TNBC. CONCLUSIONS: We identified a stemness-related gene panel associated with JQ1 and describe how this inhibitor modifies the stemness landscape in TNBC. Therefore, we propose a novel role for JQ1 as a stemness-targeting drug. Loss of the stem cell phenotype via JQ1 treatment could lead to less aggressive and more chemo-sensitive tumours, reflecting a better patient prognosis. Thus, the identified gene panel may be of interest for the clinical management of patients with aggressive TNBC.

Pharmacological screening and transcriptomic functional analyses identify a synergistic interaction between dasatinib and olaparib in triple-negative breast cancer.

Identification of druggable vulnerabilities is a main objective in triple-negative breast cancer (TNBC), where no curative therapies exist. Gene set enrichment analyses (GSEA) and a pharmacological evaluation using a library of compounds were used to select potential druggable combinations. MTT and studies with semi-solid media were performed to explore the activity of the combinations. TNBC cell lines (MDAMB-231, BT549, HS-578T and HCC3153) and an additional panel of 16 cell lines were used to assess the activity of the two compounds. Flow cytometry experiments and biochemical studies were also performed to explore the mechanism of action. GSEA were performed using several data sets (GSE21422, GSE26910, GSE3744, GSE65194 and GSE42568), and more than 35 compounds against the identified functions were evaluated to discover druggable opportunities. Analyses done with the Chou and Talalay algorithm confirmed the synergy of dasatinib and olaparib. The combination of both agents significantly induced apoptosis in a caspase-dependent manner and revealed a pleotropic effect on cell cycle: Dasatinib arrested cells in G0/G1 and olaparib in G2/M. Dasatinib inhibited pChk1 and induced DNA damage measured by pH2AX, and olaparib increased pH3. Finally, the effect of the combination was also evaluated in a panel of 18 cell lines representative of the most frequent solid tumours, observing a particularly synergism in ovarian cancer. Breast cancer, triple negative, dasatinib, olaparib, screening.

Expression of MHC class I, HLA-A and HLA-B identifies immune-activated breast tumors with favorable outcome.

Antigen recognition by MHC class I molecules is a key step for the initiation of the immune response. We hypothesized that expression of these molecules could be a marker of immune-activated breast cancers. Data from KM Plotter were extracted to develop an exploratory cohort. Information from Cancer Genome Atlas (TCGA) and METABRIC was used to create two validation cohorts. Raw data were re-processed and analyzed using plyr R and Bioconductor. We predicted epitope-HLA binding to MHC I molecules by using NetMHC 4.0. Cox proportional hazards regression was computed to correlate gene expression and survival outcome. There was a weak but positive correlation between mutational burden and the expression of most MHC class I molecules. In the exploratory cohort, expression of HLA-A and HLA-B was associated with favorable relapse-free survival (RFS) and overall survival (OS) in the basal-like subgroup. This was confirmed in the METABRIC and TCGA dataset. Expression of HLA-A and HLA-B was associated with biomarkers of T cell activation (GZMA, GZMB, and PRF1) and improved the predictive capacity of known immunologic signatures. Several neopeptides expressed in breast cancer were also identified including FUK, SNAPC3, GC, ANO8, DOT1L, HIST1H3F, MYBPH, STX2, FRMD6, CPSF1, or SMTN, among others. Expression of HLA A and B is associated with T cell activation and identifies immune activated, basal-like breast cancers with favorable prognosis. Antigen recognition markers should be incorporated into the assessment of the tumor immune state of basal-like breast patients.

Activity of BET-proteolysis targeting chimeric (PROTAC) compounds in triple negative breast cancer.

BACKGROUND: Triple negative breast cancer (TNBC) is an incurable disease where novel therapeutic strategies are needed. Proteolysis targeting chimeric (PROTAC) are novel compounds that promote protein degradation by binding to an ubiquitin ligase. In this work, we explored the antitumoral activity of two novel BET-PROTACs, MZ1 and ARV-825, in TNBC, ovarian cancer and in a BET inhibitor resistant model. METHODS: OVCAR3, SKOV3, BT549, MDA-MB-231 cell lines and the JQ1 resistant cell line MDA-MB-231R were evaluated. MTTs, colony-forming assay, three-dimensional cultures in matrigel, flow cytometry, and western blots were performed to explore the anti-proliferative effect and biochemical mechanism of action of MZ1 and ARV-825. In vivo studies included BALB/c nu/nu mice engrafted with MDA-MB-231R cells. RESULTS: The BET-PROTACs MZ1 and ARV-825 efficiently downregulated the protein expression levels of the BET protein BRD4, in MDA-MB-231 and MDA-MB-231R. MZ1 and ARV-825 also showed an antiproliferative effect on sensitive and resistant cells. This effect was corroborated in other triple negative (BT549) and ovarian cancer (SKOV3, OVCAR3) cell lines. MZ1 provoked G2/M arrest in MDA-MB-231. In addition, a profound effect on caspase-dependent apoptosis was observed in both sensitive and resistant cells. No synergistic activity was observed when it was combined with docetaxel, cisplatin or olaparib. Finally, in vivo administration of MZ1 rescued tumor growth in a JQ1-resistant xenograft model, reducing the expression levels of BRD4. CONCLUSIONS: Using both in vitro and in vivo approaches, we describe the profound activity of BET-PROTACs in parental and BETi-resistant TNBC models. This data provides options for further clinical development of these agents in TNBC.

Poly(Cyclohexene Phthalate) Nanoparticles for Controlled Dasatinib Delivery in Breast Cancer Therapy.

The effect on the activity in breast cancer models of the small tyrosine kinase inhibitor dasatinib (DAS), either alone or in combination with other antitumoral agents, has been recently explored. However, DAS is characterized by its low and highly pH-dependent solubility, which could lead to poor uptake of the drug limiting its tumoral efficacy. Thus far, the development of safe and efficient delivery vehicles of DAS to improve the therapeutic efficacy minimizing the toxicity profile is still required. In this work, a biodegradable and biocompatible polyester is assessed, for the first time, as raw material for the generation of polymeric nanoparticles (NPs). NPs of 100 nm with a narrow polydispersity were formulated for the encapsulation of DAS. The enzymatic and cellular degradation of the new drug delivery system has been studied, and the toxicity and blood compatibility evaluated for its potential clinical use. The new material used for the generation of nanoparticles led to encapsulate DAS in an efficient manner with quicker release DAS profile when compared with the FDA-approved biopolymer Polylactide. The new DAS-loaded polymeric nanocarrier gave a superior efficacy when compared to free DAS with no difference in the mechanism of action. The new NPs shown to be a promising DAS delivery system to be further evaluated for breast cancer treatment.

Screening and Preliminary Biochemical and Biological Studies of [RuCl(p-cymene)(N,N-bis(diphenylphosphino)-isopropylamine)][BF4] in Breast Cancer Models.

Breast cancer is the second leading cause of cancer death worldwide. Despite progress in drug discovery, identification of the correct population is the limiting factor to develop new compounds in the clinical setting. Therefore, the aim of this study is to evaluate the effects of a new metallodrug, [RuCl(p-cymene)(N,N-bis(diphenylphosphino)-isopropylamine)][BF4] (pnpRu-14), as a lead pnp-Ru compound by screening and preliminary biochemical and biological studies in different breast cancer subtypes. The results show that complex pnpRu-14 is much more effective in promoting in vitro cytotoxic effects on HER2+ and RH+/HER2- breast cancer than the reference metallodrugs cisplatin, carboplatin, or RAPTA-C. It is important to highlight that pnpRu-14 shows an impressive cytotoxicity against BT474 cells. Caspase-dependent apoptosis is the mechanism of action for these compounds. In addition, treatment of SKBR3, BT474, T47D, and MCF7 cancer cells with pnpRu-14 caused an accumulation of cells in the G0/G1 phase cells. The human serum albumin, DNA, and H1 histones binding properties of the lead compound are reported. Pharmacokinetic and biodistribution studies show a quick absorption of pnpRu-14 in serum with no significant accumulation in any of the tested organs. This work provides evidence to support the preclinical and clinical development of pnpRu-14 in breast cancer.

Mapping Bromodomains in breast cancer and association with clinical outcome.

A specific family of proteins that participate in epigenetic regulation is the bromodomain (BRD) family of proteins. In this work, we aimed to explore the expression of the BRD family at a transcriptomic level in breast cancer, and its association with patient survival. mRNA level data from normal breast and tumor tissues were extracted from public datasets. Gene set enrichment analysis (GSEA) was performed to identify relevant biological functions. The KM Plotter Online tool was used to evaluate the relationship between the presence of different genes and patient clinical outcome. mRNA level data from HER2+ breast cancer patients sensible and resistant to trastuzumab were also evaluated. The BRD family was an enriched function. In HER2 positive tumors the combined analyses of BRD2, BAZ1A, TRIM33 and ZMYND8 showed a detrimental relapse free survival (RFS). Similarly, the combined analysis of BRD2, BAZ1A, PHIP, TRIM33, KMT2A, ASH1L, PBRM1, correlated with an extremely poor overall survival (OS). The prognosis was confirmed using an independent dataset from TCGA. Finally, no relation between expression of BRD genes and response to trastuzumab was observed in the HER2 population. Upregulation of some BRD genes is associated with detrimental outcome in HER2 positive tumors, regardless trastuzumab treatment.