Giuseppe Merlino

Complexity in the tumour microenvironment: Cancer associated fibroblast gene expression patterns identify both common and unique features of tumour-stroma crosstalk across cancer types

Cancer is a complex disease, driven by the accumulation of several somatic aberrations but fostered by a two-way interaction between tumour cells and the surrounding microenvironment. Cancer associated fibroblasts (CAFs) represent one of the major players in tumour-stroma crosstalk. Recent in vitro and in vivo studies, often conducted by employing high throughput approaches, have started unravelling the key pathways involved in their functional effects. This review focus on open challenges in the study of CAF properties and function, highlighting at the same time the existence of common mechanisms as well as peculiarities in different cancer types (breast, prostate and lung cancer). Although still limited by current experimental models, which are unable to deal with the full level of complexity of the tumour microenvironment, a better understanding of these mechanisms may enable the identification of new biomarkers and therapeutic targets, to improve current strategies for cancer diagnosis and treatment.

Stromal Activation by Tumor Cells: An in Vitro Study in Breast Cancer

Background: The tumor microenvironment participates in the regulation of tumor progression and influences treatment sensitivity. In breast cancer, it also may play a role in determining the fate of non-invasive lesions such as ductal carcinoma in situ (DCIS), a non-obligate precursor of invasive diseases, which is aggressively treated despite its indolent nature in many patients since no biomarkers are available to predict the progression of DCIS to invasive disease. In vitro models of stromal activation by breast tumor cells might provide clues as to specific stromal genes crucial for the transition from DCIS to invasive disease. Methods: normal human dermal fibroblasts (NHDF) were treated under serum-free conditions with cell culture media conditioned by breast cancer cell lines (SkBr3, MDA-MB-468, T47D) for 72 h and subjected to gene expression profiling with Illumina platform. Results: TGM2, coding for a tissue transglutaminase, was identified as candidate gene for stromal activation. In public transcriptomic datasets of invasive breast tumors TGM2 expression proved to provide prognostic information. Conversely, its role as an early biosensor of tumor invasiveness needs to be further investigated by in situ analyses. Conclusion: Stromal TGM2 might probably be associated with precancerous evolution at earlier stages compared to DCIS.

In-depth characterization of breast cancer tumor-promoting cell transcriptome by RNA sequencing and microarrays

Numerous studies have reported the existence of tumor-promoting cells (TPC) with self-renewal potential and a relevant role in drug resistance. However, pathways and modifications involved in the maintenance of such tumor subpopulations are still only partially understood. Sequencing-based approaches offer the opportunity for a detailed study of TPC including their transcriptome modulation. Using microarrays and RNA sequencing approaches, we compared the transcriptional profiles of parental MCF7 breast cancer cells with MCF7-derived TPC (i.e. MCFS). Data were explored using different bioinformatic approaches, and major findings were experimentally validated. The different analytical pipelines (Lifescope and Cufflinks based) yielded similar although not identical results. RNA sequencing data partially overlapped microarray results and displayed a higher dynamic range, although overall the two approaches concordantly predicted pathway modifications. Several biological functions were altered in TPC, ranging from production of inflammatory cytokines (i.e., IL-8 and MCP-1) to proliferation and response to steroid hormones. More than 300 non-coding RNAs were defined as differentially expressed, and 2,471 potential splicing events were identified. A consensus signature of genes up-regulated in TPC was derived and was found to be significantly associated with insensitivity to fulvestrant in a public breast cancer patient dataset. Overall, we obtained a detailed portrait of the transcriptome of a breast cancer TPC line, highlighted the role of non-coding RNAs and differential splicing, and identified a gene signature with a potential as a context-specific biomarker in patients receiving endocrine treatment.

Extracellular matrix proteins as diagnostic markers of breast carcinoma

Changes in amount and composition of extracellular matrix (ECM) are considered a hallmark of tumor development. We tested the hypothesis that abnormal production of ECM components leads to blood‐released ECM molecules representing tumor circulating biomarkers. Candidate genes were selected through class comparison in two publicly available datasets and confirmed in paired normal and tumor associated fibroblasts from breast carcinoma (BC) specimens. Production and release of ECM molecules were evaluated in normal human dermal fibroblasts (NHDFs) treated with conditioned media from three BC cell lines. Plasma samples from healthy donors and from patients with malignant or benign breast disease were tested by ELISA for the presence of collagen 11a1 (COL11A1), collagen oligomeric matrix protein (COMP), and collagen 10a1 (COL10A1). Selected ECM molecules were investigated by IHC in malignant and benign specimens. In silico analysis of gene expression profiles identified 11 ECM genes significantly up‐regulated in tumor versus normal tissue. Western blot analyses revealed increased levels of molecules encoded by three of these genes, COL11A1, COMP, and COL10A1, in cell lysates and supernatants of conditioned NHDFs. Class comparison and class prediction analyses of two independent series of human plasma samples identified the combination of COL11A1, COMP, and COL10A1 as potentially informative in discriminating BC patients from those with benign disease. The three molecules resulted expressed in the stroma of BC tissue samples. Our results indicate that circulating COL11A1, COMP, and COL10A1 may be useful in diagnostic assessment of suspicious breast nodules and ECM molecules could represent an avenue to biomarker identification.

Prognostic and functional role of subtype‐specific tumor–stroma interaction in breast cancer

None of the clinically relevant gene expression signatures available for breast cancer were specifically developed to capture the influence of the microenvironment on tumor cells. Here, we attempted to build subtype‐specific signatures derived from an in vitro model reproducing tumor cell modifications after interaction with activated or normal stromal cells. Gene expression signatures derived from HER2+, luminal, and basal breast cancer cell lines (treated by normal fibroblasts or cancer‐associated fibroblasts conditioned media) were evaluated in clinical tumors by in silico analysis on published gene expression profiles (GEPs). Patients were classified as microenvironment‐positive (μENV+ve), that is, with tumors showing molecular profiles suggesting activation by the stroma, or microenvironment‐negative (μENV−ve) based on correlation of their tumors GEP with the respective subtype‐specific signature. Patients with estrogen receptor alpha (ER)+/HER2−/μENV+ve tumors were characterized by 2.5‐fold higher risk of developing distant metastases (HR = 2.546; 95% CI: 1.751–3.701, P = 9.84E‐07), while μENV status did not affect, or only suggested the risk of distant metastases, in women with HER2+ (HR = 1.541; 95% CI: 0.788–3.012, P = 0.206) or ER‐/HER2− tumors (HR = 1.894; 95% CI: 0.938–3.824; P = 0.0747), respectively. In ER+/HER2− tumors, the μENV status remained significantly associated with metastatic progression (HR = 2.098; CI: 1.214–3.624; P = 0.00791) in multivariable analysis including size, age, and Genomic Grade Index. Validity of our in vitro model was also supported by in vitro biological endpoints such as cell growth (MTT assay) and migration/invasion (Transwell assay). In vitro‐derived gene signatures tracing the bidirectional interaction with cancer activated fibroblasts are subtype‐specific and add independent prognostic information to classical prognostic variables in women with ER+/HER2− tumors.

Abstract 2630: MEN1309, a novel antibody drug conjugate (ADC) targeting Ly75 antigen, induces complete responses in several xenografts of solid tumors

The cell surface antigen Lymphocyte antigen 75 (LY75, CD205, DEC-205) is over-expressed in several tumor histotypes. It is a type I C-type lectin receptor (CLR), normally expressed on various APC subsets, characterized by a cytoplasmic domain containing protein motifs crucial for endocytosis and internalization upon ligation. These features make the antigen ideal to be exploited as a target for a novel ADC. MEN1309 is a humanized IgG1 antibody directed against the cell surface antigen Ly75, conjugated through a cleavable linker to a potent maytansinoid microtubule disruptor, DM4. In this study, we evaluated the in vitro and in vivo (xenografts and PDX) efficacy of MEN1309 in different tumor histotypes. A PK/PD relationship was also investigated in tumor-bearing mice. IHC demonstrated high prevalence of Ly75 in human pancreatic, triple negative breast, and bladder cancers, as well as in diffuse large B-cell lymphoma. In vitro experiments showed that cytotoxic activity of MEN1309 was in nM/sub nM range against several lymphoma, pancreatic, bladder and triple-negative breast cancer (TNBC) cell lines. Moreover, MEN1309 exhibited high cell-killing ability against cells having either strong as well as low to moderate antigen expression. In vivo, MEN1309 at 2.5-5 mg/kg (schedule varying from single dose, q7dx3, or q21dx3) showed an impressive antitumor activity, resulting in complete and long lasting responses in most of the xenograft models representing lymphoma, TNBC, bladder and pancreatic cancers, expressing the antigen at high but also at low levels. No treatment related toxicity in terms of change of body weight and death events were detected. Moreover, the administration of (i) isotype control-DM4, (ii) the non-conjugate antibody IgG1 and (iii) the free toxin DM4 (at a dosage corresponding to the equimolar concentration linked at 10 mg/kg ADC) showed little to no therapeutic efficacy on tumor growth. In TNBC patient-derived xenograft (PDX) model (coming from a heavily pre-treated patient and expressing high level of the antigen Ly75), MEN1309 (5 mg/kg q21dx3) showed a complete tumor regression. Finally, in the pancreatic adenocarcinoma xenograft model HPAFII, the pharmacokinetics profile in serum of MEN1309 at 5 mg/kg was characterized and it was qualitatively correlated, using immunofluorescence, with the occurrence of phosphorylation of Serine 10 of H3 Histone in cancer cells, as a pharmacodynamic (PD) marker of DM4 activity on microtubules. Initial ADC exposure was noteworthy and was followed by a relatively fast decline. In parallel with the decay of the serum ADC concentrations there was a progressive increase in the number of positive cells showing the PD marker for mitotic arrest. Overall, our data suggest that MEN1309 is a selective and potent novel antitumoral ADC and it deserves to enter into aPhase I study for a variety of Ly75 positive tumor histotypes. Citation Format: Mario Bigioni, Giuseppe Merlino, Cristina Bernado Morales, Rossana Bugianesi, Attilio Crea, Rosanna Manno, Joaquin Arribas, Rachel Dusek, Nickolas Attanasio, Keith Wilson, Christian Rohlff, Monica Binaschi. MEN1309, a novel antibody drug conjugate (ADC) targeting Ly75 antigen, induces complete responses in several xenografts of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2630. doi:10.1158/1538-7445.AM2017-2630

Abstract 3646: Characterization of the novel antibody drug conjugate MEN1309 and its target antigen Ly75

Ly75 (CD205, DEC-205) is a type I transmembrane glycoprotein and a C-type lectin receptor involved in antigen uptake and processing, mainly expressed by antigen presenting cells (APC). The short cytoplasmic tail contains motifs for amino acid-based endocytosis, making this receptor an ideal target antigen for an antibody drug conjugate (ADC)-based antitumoral therapy. MEN1309 is a novel fully humanized ADC which binds to Ly75 with high affinity as shown by ELISA and FACS analysis. The antibody is conjugated to a maytansinoid DM4, a potent tubulin inhibitor, through a cleavable linker.The ability of Ly75 to internalize the antibody after binding was determined using an immunoflourescence assay that showed a rapid, efficient, and near complete internalization over a one hour time course.The expression of Ly75 mRNA and protein was investigated in human cancer cell lines derived from different histotypes and revealed high expression in pancreas, bladder, triple negative breast cancer (TNBC) cells and in diffuse large B-cell lymphoma (DLBCL). Indeed, MEN1309 shows a powerful (pM range) in vitro cytotoxic activity on different cancer cell lines expressing Ly75, whereas it exerts a weaker effect on antigen-negative cells. Besides the mechanism of action (MoA) of MEN1309 as an ADC, the putative efficacy of the antibody to drive an ADCC response was investigated through in vitro binding and functional assays. In spite of a high binding affinity of MEN1309 to FcγRIIIa, no ADCC response was observed, suggesting that the high internalization rate of the antigen could hamper the triggering of NK responses.Moreover, in order to characterize the functional role of Ly75 in cancer cell lines, its expression was downregulated by siRNA demonstrating an inhibition of the proliferation rate in cells from different histotypes.Finally, we investigated if some cancer cell lines could show a higher expression of two intergenically spliced forms derived from Ly75 and DCL-1 genes recently reported in literature. We found that the intergenically spliced forms were expressed on average 30 fold less than CD205 mRNA in all the cancer cell lines analyzed, suggesting that these variants derive just from an intergenic readthrough without a specific transcriptional regulation. Citation Format: Alessandro Bressan, Alessio Fiascarelli, Giuseppe Merlino, Corrado Carrisi, Daniela Bellarosa, Rachel Dusek, Rahel Awdew, Sudha Swaminathan, Arnima Bisht, To Uyen T. Do, San Lin Lou, Dee Aud, Jonathan Terrett, Keith Wilson, Christian Rohlff, Monica Binaschi. Characterization of the novel antibody drug conjugate MEN1309 and its target antigen Ly75 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3646. doi:10.1158/1538-7445.AM2017-3646

Abstract 1701: The novel anti-CD205 antibody drug conjugate (ADC) MEN1309 shows strong antitumoral activity in diffuse large B cell lymphoma (DLBCL)

Background. Up to one third of DLBCL patients still succumb to their disease: novel therapeutic approaches are needed. MEN1309 is a novel ADC consisting of an anti-CD205 monoclonal antibody conjugated to the DM4 maytansine derivative through a cleavable linker. Here, we assessed its anti-DLBCL antitumoral activity. Methods 24 DLBCL cell lines were exposed to MEN1309 and, as control, to IgG-conjugated DM4 for 72h. Cell proliferation was measured with MTT. Apoptosis activation, defined by at least 1.5 fold increase in the caspase 3/7 signal respect to controls, was measured with the Promega ApoTox-Glo Triplex Assay. Xenografts (15 x 106 cells/mouse, 200 μL of PBS) were established s.c. into the left flanks of female NOD-SCID mice; i.v. treatments started with tumors of 250-350 mm3 volume. Results. MEN1309 showed strong cytotoxic activity in DLBCL (median IC50 = 300 pM; 95%CI, 200-771), while the IgG-conjugated DM4 toxin was 100x less active (30 nM; 95%CI, 20-33). MEN1309 induced apoptosis in 17/24 (71%) DLBCL. No difference was seen based upon DLBCL cell of origin, MYC or BCL2 translocations or TP53 status. MEN1309 activity was highly correlated with its target expression with an inverse correlation between IC50 values and CD205 expression at flow cytometry or RT-PCR (R = - 0.79, P Conclusions. In DLBCL models, the novel ADC MEN1309 had strong in vitro and in vivo anti-tumor activity, which is highly correlated with the expression of its target. Citation Format: Eugenio Gaudio, Chiara Tarantelli, Francesca Guidetti, Maurilio Ponzoni, Roberta Pittau Bordone, Alessio Fiascarelli, Andrea Rinaldi, Ivo Kwee, Afua Adjeiwaa Mensah, Anastasios Stathis, Davide Rossi, Georg Stussi, Emanuele Zucca, Giuseppe Merlino, Mario Bigioni, Monica Binaschi, Francesco Bertoni. The novel anti-CD205 antibody drug conjugate (ADC) MEN1309 shows strong antitumoral activity in diffuse large B cell lymphoma (DLBCL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1701. doi:10.1158/1538-7445.AM2017-1701