T-reg transcriptomic signatures identify response to check-point inhibitors.

Regulatory T cells (Tregs) is a subtype of CD4+ T cells that produce an inhibitory action against effector cells. In the present work we interrogated genomic datasets to explore the transcriptomic profile of breast tumors with high expression of Tregs. Only 0.5% of the total transcriptome correlated with the presence of Tregs and only four transcripts, BIRC6, MAP3K2, USP4 and SMG1, were commonly shared among the different breast cancer subtypes. The combination of these genes predicted favorable outcome, and better prognosis in patients treated with checkpoint inhibitors. Twelve up-regulated genes coded for proteins expressed at the cell membrane that included functions related to neutrophil activation and regulation of macrophages. A positive association between MSR1 and CD80 with macrophages in basal-like tumors and between OLR1, ABCA1, ITGAV, CLEC5A and CD80 and macrophages in HER2 positive tumors was observed. Expression of some of the identified genes correlated with favorable outcome and response to checkpoint inhibitors: MSR1, CD80, OLR1, ABCA1, TMEM245, and ATP13A3 predicted outcome to anti PD(L)1 therapies, and MSR1, CD80, OLR1, ANO6, ABCA1, TMEM245, and ATP13A3 to anti CTLA4 therapies, including a subgroup of melanoma treated patients. In this article we provide evidence of genes strongly associated with the presence of Tregs that modulates the response to check point inhibitors.

Latent-Transforming Growth Factor ß-Binding Protein 1/Transforming Growth Factor ß1 Complex Drives Antitumoral Effects upon ERK5 Targeting in Melanoma.

Melanoma is the deadliest skin cancer, with a poor prognosis in advanced stages. Available treatments have improved survival, although long-term benefits still are unsatisfactory. The mitogen-activated protein kinase extracellular signal-regulated kinase 5 (ERK5) promotes melanoma growth, and ERK5 inhibition determines cellular senescence and the senescence-associated secretory phenotype. Here, latent-transforming growth factor ß-binding protein 1 (LTBP1) mRNA was found to be up-regulated in A375 and SK-Mel-5 BRAFV600E melanoma cells after ERK5 inhibition. In keeping with a key role of LTBP1 in regulating transforming growth factor ß (TGF-ß), TGF-ß1 protein levels were increased in lysates and conditioned media of ERK5-knockdown (KD) cells, and were reduced upon LTBP1 KD. Both LTBP1 and TGF-ß1 proteins were increased in melanoma xenografts in mice treated with the ERK5 inhibitor XMD8-92. Moreover, treatment with conditioned media from ERK5-KD melanoma cells reduced cell proliferation and invasiveness, and TGF-ß1-neutralizing antibodies impaired these effects. In silico data sets revealed that higher expression levels of both LTBP1 and TGFB1 mRNA are associated with better overall survival of melanoma patients, and that increased LTBP1 or TGF-ß1 expression proved a beneficial role in patients treated with anti-PD1 immunotherapy, making a possible immunosuppressive role of LTBP1/TGF-ß1 unlikely upon ERK5 inhibition. This study, therefore, identifies additional desirable effects of ERK5 targeting, providing evidence of an ERK5-dependent tumor-suppressive role of TGF-ß in melanoma.

Trastuzumab deruxtecan in breast cancer.

Trastuzumab deruxtecan (T-DXd) is an antibody-drug conjugate (ADC) consisting of a humanised, anti-human epidermal growth factor receptor 2 (HER2) monoclonal antibody covalently linked to a topoisomerase I inhibitor cytotoxic payload (DXd). The high drug-to-antibody ratio (8:1) ensures a high DXd concentration is delivered to target tumour cells, following internalisation of T-DXd and subsequent cleavage of its tetrapeptide-based linker. DXd's membrane-permeable nature enables it to cross cell membranes and potentially exert antitumour activity on surrounding tumour cells regardless of HER2 expression. T-DXd's unique mechanism of action is reflected in its efficacy in clinical trials in patients with HER2-positive advanced breast cancer (in heavily pretreated populations and in those previously treated with a taxane and trastuzumab), as well as HER2-low metastatic breast cancer. Thus, ADCs such as T-DXd have the potential to change the treatment paradigm of targeting HER2 in metastatic breast cancer, including eventually within the adjuvant/neoadjuvant setting.

Synergistic effects of medicinal plants in combination with spices from algeria: Anticancer, antiangiogenic activities, and embrytoxicity studies.

ETHNOPHARMACOLOGICAL RELEVANCE: Bryonia dioica Jacq., Evernia prunastri (L.) Ach., Telephium imperati L., and Aristolochia longa L. are species widely used in traditional medicine to treat several diseases including cancer. Conjugation of two or more extracts is an approach to improve the effectiveness of their pharmacological activities. AIM OF THE STUDY: To evaluate the synergistic anticancer and anti-angiogenic effects of medicinal plants and edible species combinations. MATERIALS AND METHODS: In this work, B. dioica, E. prunastri, Telephium imperati, and Aristolochia longa extracts were conjugated to form four mixtures. The antiproliferative effect of mixtures on several carcinoma cells was examined by MTT assay, and the antiangiogenic activity was estimated through Hen's egg test in vivo. Moreover, in an Ovo model, 35 fertilized Ross eggs were used to test the embryotoxicity of mixtures. RESULTS: At the highest concentration of 200 µg/mL, both mixtures exerted an important cytotoxic effect against human carcinoma cells. The mixture BETE (Bryonia Evernia Telephium Extract) significantly reduced HT-29, PC-3, and A-549 cell viability. Likewise, this mixture strongly suppressed vascularization in vivo at 200 µg/mL. Interestingly, no signs of toxicity on Perdix embryos were recorded within 21 days of treatment. More importantly, the mixture did not have any cytotoxic effect on non cancerous cells. CONCLUSION: Taken together, our results suggest that the synergy between B. dioica, E. prunastri and T. imperati may be promising for developing new anti-cancer treatments.

ERK5 Interacts with Mitochondrial Glutaminase and Regulates Its Expression.

Many of the biological processes of the cell, from its structure to signal transduction, involve protein-protein interactions. On this basis, our aim was to identify cellular proteins that interact with ERK5, a serine/threonine protein kinase with a key role in tumor genesis and progression and a promising therapeutic target in many tumor types. Using affinity chromatography, immunoprecipitation, and mass spectrometry techniques, we unveiled an interaction between ERK5 and the mitochondrial glutaminase GLS in pancreatic tumor cells. Subsequent co-immunoprecipitation and immunofluorescence studies supported this interaction in breast and lung tumor cells as well. Genetic approaches using RNA interference techniques and CRISPR/Cas9 technology demonstrated that the loss of ERK5 function led to increased protein levels of GLS isoforms (KGA/GAC) and a concomitant increase in their activity in tumor cells. It is well known that the tumor cell reprograms its intermediary metabolism to meet its increased metabolic needs. In this sense, mitochondrial GLS is involved in the first step of glutamine catabolism, one of the main energy sources in the context of cancer. Our data suggest that ERK5 contributes to the regulation of tumor cell energy metabolism via glutaminolysis.

Considerations for the clinical development of immuno-oncology agents in cancer.

Targeting of the immune system has shown to be a successful therapeutic approach in cancer, with the development of check point inhibitors (ICI) or T-cell engagers (TCE). As immuno-oncology agents modulate the immune system to attack cancer cells and do not act directly on oncogenic vulnerabilities, specific characteristics of these compounds should be taken in consideration during clinical development. In this review we will discuss relevant concepts including limitations of preclinical models, special pharmacologic boundaries, clinical development strategies such as the selection of clinical indication, line of treatment and backbone partner, as well as the endpoints and expected magnitude of benefit required at different stages of the drug development. In addition, future directions for early and late trial designs will be reviewed. Examples from approved drugs or those currently in clinical development will be discussed and options to overcome these limitations will be provided.

An amino acid transporter subunit as an antibody-drug conjugate target in colorectal cancer.

BACKGROUND: Advanced colorectal cancer (CRC) is difficult to treat. For that reason, the development of novel therapeutics is necessary. Here we describe a potentially actionable plasma membrane target, the amino acid transporter protein subunit CD98hc. METHODS: Western blot and immunohistochemical analyses of CD98hc protein expression were carried out on paired normal and tumoral tissues from patients with CRC. Immunofluorescence and western studies were used to characterize the action of a DM1-based CD98hc-directed antibody-drug conjugate (ADC). MTT and Annexin V studies were performed to evaluate the effect of the anti-CD98hc-ADC on cell proliferation and apoptosis. CRISPR/Cas9 and shRNA were used to explore the specificity of the ADC. In vitro analyses of the antitumoral activity of the anti-CD98hc-ADC on 3D patient-derived normal as well as tumoral organoids were also carried out. Xenografted CRC cells and a PDX were used to analyze the antitumoral properties of the anti-CD98hc-ADC. RESULTS: Genomic as well proteomic analyses of paired normal and tumoral samples showed that CD98hc expression was significantly higher in tumoral tissues as compared to levels of CD98hc present in the normal colonic tissue. In human CRC cell lines, an ADC that recognized the CD98hc ectodomain, reached the lysosomes and exerted potent antitumoral activity. The specificity of the CD98hc-directed ADC was demonstrated using CRC cells in which CD98hc was decreased by shRNA or deleted using CRISPR/Cas9. Studies in patient-derived organoids verified the antitumoral action of the anti-CD98hc-ADC, which largely spared normal tissue-derived colon organoids. In vivo studies using xenografted CRC cells or patient-derived xenografts confirmed the antitumoral activity of the anti-CD98hc-ADC. CONCLUSIONS: The studies herewith reported indicate that CD98hc may represent a novel ADC target that, upon well-designed clinical trials, could be used to increase the therapeutic armamentarium against CRC.

Etiopathogenic role of ERK5 signaling in sarcoma: prognostic and therapeutic implications.

Sarcomas constitute a heterogeneous group of rare and difficult-to-treat tumors that can affect people of all ages, representing one of the most common forms of cancer in childhood and adolescence. Little is known about the molecular entities involved in sarcomagenesis. Therefore, the identification of processes that lead to the development of the disease may uncover novel therapeutic opportunities. Here, we show that the MEK5/ERK5 signaling pathway plays a critical role in the pathogenesis of sarcomas. By developing a mouse model engineered to express a constitutively active form of MEK5, we demonstrate that the exclusive activation of the MEK5/ERK5 pathway can promote sarcomagenesis. Histopathological analyses identified these tumors as undifferentiated pleomorphic sarcomas. Bioinformatic studies revealed that sarcomas are the tumors in which ERK5 is most frequently amplified and overexpressed. Moreover, analysis of the impact of ERK5 protein expression on overall survival in patients diagnosed with different sarcoma types in our local hospital showed a 5-fold decrease in median survival in patients with elevated ERK5 expression compared with those with low expression. Pharmacological and genetic studies revealed that targeting the MEK5/ERK5 pathway drastically affects the proliferation of human sarcoma cells and tumor growth. Interestingly, sarcoma cells with knockout of ERK5 or MEK5 were unable to form tumors when engrafted into mice. Taken together, our results reveal a role of the MEK5/ERK5 pathway in sarcomagenesis and open a new scenario to be considered in the treatment of patients with sarcoma in which the ERK5 pathway is pathophysiologically involved.

Chemical-proteomics Identify Peroxiredoxin-1 as an Actionable Target in Triple-negative Breast Cancer.

Triple-negative breast cancer (TNBC) is difficult to treat; therefore, the development of drugs directed against its oncogenic vulnerabilities is a desirable goal. Herein, we report the antitumor effects of CM728, a novel quinone-fused oxazepine, against this malignancy. CM728 potently inhibited TNBC cell viability and decreased the growth of MDA-MB-231-induced orthotopic tumors. Furthermore, CM728 exerted a strong synergistic antiproliferative effect with docetaxel in vitro and this combination was more effective than the individual treatments in vivo. Chemical proteomic approaches revealed that CM728 bound to peroxiredoxin-1 (Prdx1), thereby inducing its oxidation. Molecular docking corroborated these findings. CM728 induced oxidative stress and a multi-signal response, including JNK/p38 MAPK activation and STAT3 inhibition. Interestingly, Prdx1 downregulation mimicked these effects. Finally, CM728 led to DNA damage, cell cycle blockage at the S and G2/M phases, and the activation of caspase-dependent apoptosis. Taken together, our results identify a novel compound with antitumoral properties against TNBC. In addition, we describe the mechanism of action of this drug and provide a rationale for the use of Prdx1 inhibitors, such as CM728, alone or in combination with other drugs, for the treatment of TNBC.

The WNK1-ERK5 route plays a pathophysiological role in ovarian cancer and limits therapeutic efficacy of trametinib.

BACKGROUND: The dismal prognosis of advanced ovarian cancer calls for the development of novel therapies to improve disease outcome. In this regard, we set out to discover new molecular entities and to assess the preclinical effectiveness of their targeting. METHODS: Cell lines, mice and human ovarian cancer samples were used. Proteome profiling of human phosphokinases, in silico genomic analyses, genetic (shRNA and CRISPR/Cas9) and pharmacological strategies as well as an ex vivo human preclinical model were performed. RESULTS: We identified WNK1 as a highly phosphorylated protein in ovarian cancer and found that its activation or high expression had a negative impact on patients' survival. Genomic analyses showed amplification of WNK1 in human ovarian tumours. Mechanistically, we demonstrate that WNK1 exerted its action through the MEK5-ERK5 signalling module in ovarian cancer. Loss of function, genetic or pharmacological experiments, demonstrated anti-proliferative and anti-tumoural effects of the targeting of the WNK1-MEK5-ERK5 route. Additional studies showed that this pathway modulated the anti-tumoural properties of the MEK1/2 inhibitor trametinib. Thus, treatment with trametinib activated the WNK1-MEK5-ERK5 route, raising the possibility that this effect may limit the therapeutic benefit of ERK1/2 targeting in ovarian cancer. Moreover, in different experimental settings, including an ex vivo patient-derived model consisting of ovarian cancer cells cultured with autologous patient sera, we show that inhibition of WNK1 or MEK5 increased the anti-proliferative and anti-tumour efficacy of trametinib. CONCLUSIONS: The present study uncovers the participation of WNK1-MEK5-ERK5 axis in ovarian cancer pathophysiology, opening the possibility of acting on this pathway with therapeutic purposes. Another important finding of the present study was the activation of that signalling axis by trametinib, bypassing the anti-tumoural efficacy of this drug. That fact should be considered in the context of the use of trametinib in ovarian cancer.

An anti-EGFR antibody-drug conjugate overcomes resistance to HER2-targeted drugs.

Trastuzumab-emtansine (T-DM1) is an antibody-drug conjugate (ADC) that was approved in 2013 to treat HER2+ breast cancer. Despite its efficacy in the clinic, some patients exhibit intrinsic or acquired resistance to such ADC. To characterize mechanisms of resistance to T-DM1, we isolated several HER2+ resistant clones derived from the HCC1954 HER2+ cell line. The isolated clones were different as per their transcriptomic profiles. However, all the T-DM1-resistant clones showed decreased HER2 levels. Yet, the clones were still oncogenically dependent on HER2, as indicated by knock down experiments. The decrease in HER2 expression caused acquired resistance to T-DM1 and to other anti-HER2 therapies. Antibody array analyses showed that the epidermal growth factor receptor (EGFR) was expressed in these T-DM1-resistant HCC1954 clones. Indeed, therapies targeting EGFR, particularly cetuximab-DM1, demonstrated a strong anti-proliferative action on cells with acquired resistance to T-DM1 and HER2 loss. The expression of EGFR in cells resistant to T-DM1 offers the possibility of using therapies directed to this receptor to combat resistance to anti-HER2 drugs and loss of HER2 overexpression.

Immune Checkpoint Inhibitors and RAS-ERK Pathway-Targeted Drugs as Combined Therapy for the Treatment of Melanoma.

Metastatic melanoma is a highly immunogenic tumor with very poor survival rates due to immune system escape-mechanisms. Immune checkpoint inhibitors (ICIs) targeting the cytotoxic T-lymphocyte-associated protein 4 (CTLA4) and the programmed death-1 (PD1) receptors, are being used to impede immune evasion. This immunotherapy entails an increment in the overall survival rates. However, melanoma cells respond with evasive molecular mechanisms. ERK cascade inhibitors are also used in metastatic melanoma treatment, with the RAF activity blockade being the main therapeutic approach for such purpose, and in combination with MEK inhibitors improves many parameters of clinical efficacy. Despite their efficacy in inhibiting ERK signaling, the rewiring of the melanoma cell-signaling results in disease relapse, constituting the reinstatement of ERK activation, which is a common cause of some resistance mechanisms. Recent studies revealed that the combination of RAS-ERK pathway inhibitors and ICI therapy present promising advantages for metastatic melanoma treatment. Here, we present a recompilation of the combined therapies clinically evaluated in patients.

HER3 in cancer: from the bench to the bedside.

The HER3 protein, that belongs to the ErbB/HER receptor tyrosine kinase (RTK) family, is expressed in several types of tumors. That fact, together with the role of HER3 in promoting cell proliferation, implicate that targeting HER3 may have therapeutic relevance. Furthermore, expression and activation of HER3 has been linked to resistance to drugs that target other HER receptors such as agents that act on EGFR or HER2. In addition, HER3 has been associated to resistance to some chemotherapeutic drugs. Because of those circumstances, efforts to develop and test agents targeting HER3 have been carried out. Two types of agents targeting HER3 have been developed. The most abundant are antibodies or engineered antibody derivatives that specifically recognize the extracellular region of HER3. In addition, the use of aptamers specifically interacting with HER3, vaccines or HER3-targeting siRNAs have also been developed. Here we discuss the state of the art of the preclinical and clinical development of drugs aimed at targeting HER3 with therapeutic purposes.

Therapy-Induced Senescence Enhances the Efficacy of HER2-Targeted Antibody-Drug Conjugates in Breast Cancer.

Antibody-drug conjugates (ADC) are antineoplastic agents recently introduced into the antitumor arsenal. T-DM1, a trastuzumab-based ADC that relies on lysosomal processing to release the payload, is approved for HER2-positive breast cancer. Next-generation ADCs targeting HER2, such as [vic-]trastuzumab duocarmazine (SYD985), bear linkers cleavable by lysosomal proteases and membrane-permeable drugs, mediating a bystander effect by which neighboring antigen-negative cells are eliminated. Many antitumor therapies, like DNA-damaging agents or CDK4/6 inhibitors, can induce senescence, a cellular state characterized by stable cell-cycle arrest. Another hallmark of cellular senescence is the enlargement of the lysosomal compartment. Given the relevance of the lysosome to the mechanism of action of ADCs, we hypothesized that therapies that induce senescence would potentiate the efficacy of HER2-targeting ADCs. Treatment with the DNA-damaging agent doxorubicin and CDK4/6 inhibitor induced lysosomal enlargement and senescence in several breast cancer cell lines. While senescence-inducing drugs did not increase the cytotoxic effect of ADCs on target cells, the bystander effect was enhanced when HER2-negative cells were cocultured with HER2-low cells. Knockdown experiments demonstrated the importance of cathepsin B in the enhanced bystander effect, suggesting that cathepsin B mediates linker cleavage. In breast cancer patient-derived xenografts, a combination treatment of CDK4/6 inhibitor and SYD985 showed improved antitumor effects over either treatment alone. These data support the strategy of combining next-generation ADCs targeting HER2 with senescence-inducing therapies for tumors with heterogenous and low HER2 expression. SIGNIFICANCE: Combining ADCs against HER2-positive breast cancers with therapies that induce cellular senescence may improve their therapeutic efficacy by facilitating a bystander effect against antigen-negative tumor cells.

Genomic mapping of copy number variations influencing immune response in breast cancer.

Identification of genomic alterations that influence the immune response within the tumor microenvironment is mandatory in order to identify druggable vulnerabilities. In this article, by interrogating public genomic datasets we describe copy number variations (CNV) present in breast cancer (BC) tumors and corresponding subtypes, associated with different immune populations. We identified regulatory T-cells associated with the Basal-like subtype, and type 2 T-helper cells with HER2 positive and the luminal subtype. Using gene set enrichment analysis (GSEA) for the Type 2 T-helper cells, the most relevant processes included the ERBB2 signaling pathway and the Fibroblast Growth Factor Receptor (FGFR) signaling pathway, and for CD8+ T-cells, cellular response to growth hormone stimulus or the JAK-STAT signaling pathway. Amplification of ERBB2, GRB2, GRB7, and FGF receptor genes strongly correlated with the presence of type 2 T helper cells. Finally, only 8 genes were highly upregulated and present in the cellular membrane: MILR1, ACE, DCSTAMP, SLAMF8, CD160, IL2RA, ICAM2, and SLAMF6. In summary, we described immune populations associated with genomic alterations with different BC subtypes. We observed a clear presence of inhibitory cells, like Tregs or Th2 when specific chromosomic regions were amplified in basal-like or HER2 and luminal groups. Our data support further evaluation of specific therapeutic strategies in specific BC subtypes, like those targeting Tregs in the basal-like subtype.

Enhanced Antitumoral Activity of Encapsulated BET Inhibitors When Combined with PARP Inhibitors for the Treatment of Triple-Negative Breast and Ovarian Cancers.

BRCA1/2 protein-deficient or mutated cancers comprise a group of aggressive malignancies. Although PARPis have shown considerably efficacy in their treatment, the widespread use of these agents in clinical practice is restricted by various factors, including the development of acquired resistance due to the presence of compensatory pathways. BETis can completely disrupt the HR pathway by repressing the expression of BRCA1 and could be aimed at generation combination regimes to overcome PARPi resistance and enhance PARPi efficacy. Due to the poor pharmacokinetic profile and short half-life, the first-in-class BETi JQ1 was loaded into newly developed nanocarrier formulations to improve the effectivity of olaparib for the treatment of BRCAness cancers. First, polylactide polymeric nanoparticles were generated by double emulsion. Moreover, liposomes were prepared by ethanol injection and evaporation solvent method. JQ1-loaded drug delivery systems display optimal hydrodynamic radii between 60 and 120 nm, with a very low polydispersity index (PdI), and encapsulation efficiencies of 92 and 16% for lipid- and polymeric-based formulations, respectively. Formulations show high stability and sustained release. We confirmed that all assayed JQ1 formulations improved antiproliferative activity compared to the free JQ1 in models of ovarian and breast cancers. In addition, synergistic interaction between JQ1 and JQ1-loaded nanocarriers and olaparib evidenced the ability of encapsulated JQ1 to enhance antitumoral activity of PARPis.

ERK5 Is a Major Determinant of Chemical Sarcomagenesis: Implications in Human Pathology.

Sarcomas are a heterogeneous group of tumors in which the role of ERK5 is poorly studied. To clarify the role of this MAPK in sarcomatous pathology, we used a murine 3-methyl-cholanthrene (3MC)-induced sarcoma model. Our data show that 3MC induces pleomorphic sarcomas with muscle differentiation, showing an increased expression of ERK5. Indeed, this upregulation was also observed in human sarcomas of muscular origin, such as leiomyosarcoma or rhabdomyosarcoma. Moreover, in cell lines derived from these 3MC-induced tumors, abrogation of Mapk7 expression by using specific shRNAs decreased in vitro growth and colony-forming capacity and led to a marked loss of tumor growth in vivo. In fact, transcriptomic profiling in ERK5 abrogated cell lines by RNAseq showed a deregulated gene expression pattern for key biological processes such as angiogenesis, migration, motility, etc., correlating with a better prognostic in human pathology. Finally, among the various differentially expressed genes, Klf2 is a key mediator of the biological effects of ERK5 as indicated by its specific interference, demonstrating that the ERK5-KLF2 axis is an important determinant of sarcoma biology that should be further studied in human pathology.

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.

Surfaceome analyses uncover CD98hc as an antibody drug-conjugate target in triple negative breast cancer.

BACKGROUND: Despite the incorporation of novel therapeutics, advanced triple negative breast cancer (TNBC) still represents a relevant clinical problem. Considering this, as well as the clinical efficacy of antibody-drug conjugates (ADCs), we aimed at identifying novel ADC targets that could be used to treat TNBC. METHODS: Transcriptomic analyses were performed on TNBC and normal samples from three different studies. Plasma membrane proteins of three cell lines representative of the TNBC subtype were identified by cell surface biotinylation or plasma membrane isolation, followed by analyses of cell surface proteins using the Surfaceome online tool. Immunofluorescence and western studies were used to characterize the action of a CD98hc-directed ADC, which was prepared by in house coupling of emtansine to an antibody that recognized the ectodomain of CD98hc. Xenografted TNBC cells were used to analyze the antitumoral properties of the anti-CD98hc ADC. RESULTS: Comparative genomic studies between normal breast and TNBC tissues, together with proteomic and bioinformatic analyses resulted in the elaboration of a catalog of potential ADC targets. One of them, the CD98hc transmembrane protein, was validated as an ADC target. An antibody recognizing the ectodomain of CD98hc efficiently internalized and reached the lysosomal compartment. An emtansine-based ADC derived from such antibody was prepared and showed antitumoral properties in TNBC in vitro and in vivo models. Mechanistically, the anti-CD98hc ADC blocked cell cycle progression, that was followed by cell death caused by mitotic catastrophe. CONCLUSIONS: This work describes a list of potential ADC targets in TNBC and validates one of them, the transmembrane protein CD98hc. The studies presented here also demonstrate the robustness of the multiomic approach herewith described to identify novel potential ADC targets.

mTOR Inhibition and T-DM1 in HER2-Positive Breast Cancer.

In patients with trastuzumab-resistant HER2-positive breast cancer, the combination of everolimus (mTORC1 inhibitor) with trastuzumab failed to show a clinically significant benefit. However, the combination of mTOR inhibition and the antibody-drug conjugate (ADC) trastuzumab-emtansine (T-DM1) remains unexplored. We tested T-DM1 plus everolimus in a broad panel of HER2-positive breast cancer cell lines. The combination was superior to T-DM1 alone in four cell lines (HCC1954, SKBR3, EFM192A, and MDA-MB-36) and in two cultures from primary tumor cells derived from HER2-positive patient-derived xenografts (PDX), but not in BT474 cells. In the trastuzumab-resistant HCC1954 cell line, we characterized the effects of the combination using TAK-228 (mTORC1 and -2 inhibitor) and knockdown of the different mTOR complex components. T-DM1 did not affect mTOR downstream signaling nor induct autophagy. Importantly, mTOR inhibition increased intracellular T-DM1 levels, leading to increased lysosomal accumulation of the compound. The increased efficacy of mTOR inhibition plus T-DM1 was abrogated by lysosome inhibitors (chloroquine and bafilomycin A1). Our experiments suggest that BT474 are less sensitive to T-DM1 due to lack of optimal lysosomal processing and intrinsic resistance to the DM1 moiety. Finally, we performed several in vivo experiments that corroborated the superior activity of T-DM1 and everolimus in HCC1954 and PDX-derived mouse models. In summary, everolimus in combination with T-DM1 showed strong antitumor effects in HER2-positive breast cancer, both in vitro and in vivo. This effect might be related, at least partially, to mTOR-dependent lysosomal processing of T-DM1, a finding that might apply to other ADCs that require lysosomal processing. IMPLICATIONS: Inhibition of mTOR increases the antitumor activity of T-DM1, supporting that the combination of mTOR inhibitors and antibody-drug conjugates warrants clinical evaluation in patients with HER2-positive breast cancer.