Nicolò Panini

Macrophages are essential for maintaining a M2 protective response early after ischemic brain injury

Resident microglia and recruited macrophages are major contributors to the post-ischemic inflammatory response. Initially considered functionally homogeneous populations, data now suggest distinct but still controversial roles after brain injury. Using a model of conditional monocyte/macrophage depletion we studied the contribution of these myeloid cells to brain lesion progression after ischemia, and their influence on the ischemic inflammatory environment. Male CD11b-DTR transgenic mice, expressing the human diphtheria toxin receptor under the control of the CD11b promoter, were treated with diphtheria toxin to induce monocyte/macrophage depletion. Twenty four hours later the middle cerebral artery was permanently occluded. The ischemic lesion was measured 24h after injury. At the same time microglia and macrophage activation and polarization were assessed by quantitative immunohistochemistry and confocal microscopy for CD45high, CD11b, CD68, CD16/32, iNOS, Arg1, Ym1, and CD206, and gene expression was investigated on CD11b+ sorted cells. Depletion of monocytes/macrophages worsened the ischemic lesion within 24h after the ischemic insult. This effect was associated with higher M1/M2 polarization ratio in the ischemic lesion. Moreover, depletion increased the expression of M1 phenotypic markers on CD11b positive cells. Gene expression on CD11b+ sorted cells indicated a selective increase of iNOS and lower Arg1 mRNA expression than in non depleted mice. Depletion of monocytes/macrophages increases the ischemic lesion, an effect accompanied by an increase in the M1/M2 polarization ratio of microglia and macrophages in the ischemic area. Thus in ischemic injury recruited monocytes/macrophages may control an excessive M1 pro-inflammatory response, suggesting their ability to drive M2 protective polarization.

The bromodomain inhibitor OTX015 (MK-8628) exerts anti-tumor activity in triple-negative breast cancer models as single agent and in combination with everolimus

Triple-negative breast cancer (TNBC) is an aggressive and heterogeneous subgroup of breast tumors clinically defined by the lack of estrogen, progesterone and HER2 receptors, limiting the use of the targeted therapies employed in other breast malignancies. Recent evidence indicates that c-MYC is a key driver of TNBC. The BET-bromodomain inhibitor OTX015 (MK-8628) has potent antiproliferative activity accompanied by c-MYC down-regulation in several tumor types, and has demonstrated synergism with the mTOR inhibitor everolimus in different models. The aim of this study was to evaluate the anti-tumor activity of OTX015 as single agent and in combination with everolimus in TNBC models. OTX015 was assayed in three human TNBC-derived cell lines, HCC1937, MDA-MB-231 and MDA-MB-468, all showing antiproliferative activity after 72 h (GI50 = 75–650 nM). This was accompanied by cell cycle arrest and decreased expression of cancer stem cells markers. However, c-MYC protein and mRNA levels were only down-regulated in MDA-MB-468 cells. Gene set enrichment analysis showed up-regulation of genes involved in epigenetic control of transcription, chromatin and the cell cycle, and down-regulation of stemness-related genes. In vitro, combination with everolimus was additive in HCC1937 and MDA-MB-231 cells, but antagonistic in MDA-MB-468 cells. In MDA-MB-231 murine xenografts, tumor mass was significantly (p < 0.05) reduced by OTX015 with respect to vehicle-treated animals (best T/C = 40.7%). Although everolimus alone was not active, the combination was more effective than OTX015 alone (best T/C = 20.7%). This work supports current clinical trials with OTX015 in TNBC (NCT02259114).

Antitumour activity of trabectedin in myelodysplastic/myeloproliferative neoplasms

Background:Juvenile myelomonocytic leukaemia (JMML) and chronic myelomonocytic leukaemia (CMML) are myelodysplastic myeloproliferative (MDS/MPN) neoplasms with unfavourable prognosis and without effective chemotherapy treatment. Trabectedin is a DNA minor groove binder acting as a modulator of transcription and interfering with DNA repair mechanisms; it causes selective depletion of cells of the myelomonocytic lineage. We hypothesised that trabectedin might have an antitumour effect on MDS/MPN.Methods:Malignant CD14+ monocytes and CD34+ haematopoietic progenitor cells were isolated from peripheral blood/bone marrow mononuclear cells. The inhibition of CFU-GM colonies and the apoptotic effect on CD14+ and CD34+ induced by trabectedin were evaluated. Trabectedin’s effects were also investigated in vitro on THP-1, and in vitro and in vivo on MV-4-11 cell lines.Results:On CMML/JMML cells, obtained from 20 patients with CMML and 13 patients with JMML, trabectedin – at concentration pharmacologically reasonable, 1–5 nM – strongly induced apoptosis and inhibition of growth of haematopoietic progenitors (CFU-GM). In these leukaemic cells, trabectedin downregulated the expression of genes belonging to the Rho GTPases pathway (RAS superfamily) having a critical role in cell growth and cytoskeletal dynamics. Its selective activity on myelomonocytic malignant cells was confirmed also on in vitro THP-1 cell line and on in vitro and in vivo MV-4-11 cell line models.Conclusions:Trabectedin could be good candidate for clinical studies in JMML/CMML patients.

Promising in vivo efficacy of the BET bromodomain inhibitor OTX015/MK-8628 in malignant pleural mesothelioma xenografts.

It has recently been reported that a large proportion of human malignant pleural mesothelioma (MPM) cell lines and patient tissue samples present high expression of the c‐MYC oncogene. This gene drives several tumorigenic processes and is overexpressed in many cancers. Although c‐MYC is a strategic target to restrain cancer processes, no drugs acting as c‐MYC inhibitors are available. The novel thienotriazolodiazepine small‐molecule bromodomain inhibitor OTX015/MK‐8628 has shown potent antiproliferative activity accompanied by c‐MYC downregulation in several tumor types. This study was designed to evaluate the growth inhibitory effect of OTX015 on patient‐derived MPM473, MPM487 and MPM60 mesothelioma cell lines and its antitumor activity in three patient‐derived xenograft models, MPM473, MPM487 and MPM484, comparing it with cisplatin, gemcitabine and pemetrexed, three agents which are currently used to treat MPM in the clinic. OTX015 caused a significant delay in cell growth both in vitro and in vivo. It was the most effective drug in MPM473 xenografts and showed a similar level of activity as the most efficient treatment in the other two MPM models (gemcitabine in MPM487 and cisplatin in MPM484). In vitro studies showed that OTX015 downregulated c‐MYC protein levels in both MPM473 and MPM487 cell lines. Our findings represent the first evidence of promising therapeutic activity of OTX015 in mesothelioma.

NG2, a common denominator for neuroinflammation, blood–brain barrier alteration, and oligodendrocyte precursor response in EAE, plays a role in dendritic cell activation

In adult CNS, nerve/glial-antigen 2 (NG2) is expressed by oligodendrocyte progenitor cells (OPCs) and is an early marker of pericyte activation in pathological conditions. NG2 could, therefore, play a role in experimental autoimmune encephalomyelitis (EAE), a disease associated with increased blood–brain barrier (BBB) permeability, inflammatory infiltrates, and CNS damage. We induced EAE in NG2 knock-out (NG2KO) mice and used laser confocal microscopy immunofluorescence and morphometry to dissect the effect of NG2 KO on CNS pathology. NG2KO mice developed milder EAE than their wild-type (WT) counterparts, with less intense neuropathology associated with a significant improvement in BBB stability. In contrast to WT mice, OPC numbers did not change in NG2KO mice during EAE. Through FACS and confocal microscopy, we found that NG2 was also expressed by immune cells, including T cells, macrophages, and dendritic cells (DCs). Assessment of recall T cell responses to the encephalitogen by proliferation assays and ELISA showed that, while WT and NG2KO T cells proliferated equally to the encephalitogenic peptide MOG35-55, NG2KO T cells were skewed towards a Th2-type response. Because DCs could be responsible for this effect, we assessed their expression of IL-12 by PCR and intracellular FACS. IL-12-expressing CD11c+ cells were significantly decreased in MOG35-55-primed NG2KO lymph node cells. Importantly, in WT mice, the proportion of IL-12-expressing cells was significantly lower in CD11c+ NG2- cells than in CD11c+ NG2+ cells. To assess the relevance of NG2 at immune system and CNS levels, we induced EAE in bone-marrow chimeric mice, generated with WT recipients of NG2KO bone-marrow cells and vice versa. Regardless of their original phenotype, mice receiving NG2KO bone marrow developed milder EAE than those receiving WT bone marrow. Our data suggest that NG2 plays a role in EAE not only at CNS/BBB level, but also at immune response level, impacting on DC activation and thereby their stimulation of reactive T cells, through controlling IL-12 expression.

3D Silicon Microstructures: A New Tool for Evaluating Biological Aggressiveness of Tumor Cells

In this work, silicon micromachined structures (SMS), consisting of arrays of 3- μm-thick silicon walls separated by 50- μm-deep, 5- μm-wide gaps, were applied to investigate the behavior of eight tumor cell lines, with different origins and biological aggressiveness, in a three-dimensional (3D) microenvironment. Several cell culture experiments were performed on 3D-SMS and cells grown on silicon were stained for fluorescence microscopy analyses. Most of the tumor cell lines recognized in the literature as highly aggressive (OVCAR-5, A375, MDA-MB-231, and RPMI-7951) exhibited a great ability to enter and colonize the narrow deep gaps of the SMS, whereas less aggressive cell lines (OVCAR-3, Capan-1, MCF7, and NCI-H2126) demonstrated less penetration capability and tended to remain on top of the SMS. Quantitative image analyses of several fluorescence microscopy fields of silicon samples were performed for automatic cell recognition and count, in order to quantify the fraction of cells inside the gaps, with respect to the total number of cells in the examined field. Our results show that higher fractions of cells in the gaps are obtained with more aggressive cell lines, thus supporting in a quantitative way the observation that the behavior of tumor cells on the 3D-SMS depends on their aggressiveness level.

Counteracting roles of MHCI and CD8 + T cells in the peripheral and central nervous system of ALS SOD1 G93A mice

BackgroundThe major histocompatibility complex I (MHCI) is a key molecule for the interaction of mononucleated cells with CD8+T lymphocytes. We previously showed that MHCI is upregulated in the spinal cord microglia and motor axons of transgenic SOD1G93A mice.MethodsTo assess the role of MHCI in the disease, we examined transgenic SOD1G93A mice crossbred with β2 microglobulin-deficient mice, which express little if any MHCI on the cell surface and are defective for CD8+ T cells.ResultsThe lack of MHCI and CD8+ T cells in the sciatic nerve affects the motor axon stability, anticipating the muscle atrophy and the disease onset. In contrast, MHCI depletion in resident microglia and the lack of CD8+ T cell infiltration in the spinal cord protect the cervical motor neurons delaying the paralysis of forelimbs and prolonging the survival of SOD1G93A mice.ConclusionsWe provided straightforward evidence for a dual role of MHCI in the peripheral nervous system (PNS) compared to the CNS, pointing out regional and temporal differences in the clinical responses of ALS mice. These findings offer a possible explanation for the failure of systemic immunomodulatory treatments and suggest new potential strategies to prevent the progression of ALS.

Abstract 2352: Effect of inhibition of cell cycle versus transcription cyclin-dependent kinases (CDKs) in ovarian cancer cells

Background. Cell cycle is regulated by cyclin-dependent kinases (CDKs) activity, whose deregulation can lead to uncontrolled proliferation and cancer. Other CDKs are engaged in the regulation of transcription and post-transcriptional mRNA processing through the phosphorylation of the C-terminal domain of RNA polymerase II, such as CDK9 and CDK12. Inhibitors of cell cycle CDKs have been developed as anticancer agents and some of them are under clinical validation (e.g. palbociclib). While recent data would suggest that inhibition of CDK9 is feasible and has antitumor effect, the data on the therapeutic role of CDK12 inhibition are very scanty. Methods. Ovarian cancer cell lines were maintained in RPMI medium supplemented with 5% glutamine and 10% FBS. Cells were treated with different drug concentrations and after 72 hours cell survival was evaluated by MTS assay (Promega). IC50 values were calculated by interpolation method. Cell cycle analysis and apoptosis were performed with standard flow cytometric methods. A2780 and SKOV3 ovarian cancer cell lines knocked out for CDK12 were generated with CRISPR/CAS9 genome editing tool. Results. The cytotoxicity of palbociclib (a CDK4/6 inhibitor) and LDC000067 (a CDK9 inhibitor) was tested in a panel of ovarian cancer cell lines (A2780, SKOV3, OVCAR3, OVCAR5, OVCAR8, OVCA432, OVCA433, IGROV1, EFO27). Sensitivity of cells was similar for palbociclib and LDC000067, ranging from 10 to 33 and from 8 to 60 μM, respectively. A preferential G1 block was observed with palbociclib, while LDC000067 caused a S-G2 block. A higher induction of apoptosis was observed after LDC000067 than after palbociclib treatment in both A2780 and SKOV3. The palbociclib-induced G1 block was associated with decreased Rb phosphorylation, while no modulation of the Ser2 in the carboxyterminal domain of RNA polymerase II was observed after LDC000067 treatment. We generated CDK12 knocked out cells transfecting CRISPR/CAS9 engineered plasmid in both A2780 and SKOV3 ovarian cancer lines. The biological and pharmacological characterization of these clones is under study. Conclusions. Palbociclib and LDC000067 showed a dose dependent cytotoxic effect in the panel of ovarian cancer cell lines tested and were active in the μM range. Preliminary data of treatment induced cell cycle perturbation and apoptosis suggest that the two drugs behave in a different manner and have distinct molecular effects on cells. Citation Format: Rosaria Chila, Nicolo Panini, Eugenio Erba, Giovanna Damia, Massimo Broggini. Effect of inhibition of cell cycle versus transcription cyclin-dependent kinases (CDKs) in ovarian cancer cells [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 2352. doi:10.1158/1538-7445.AM2017-2352

48 Trabectedin and lurbinectedin are effective against leukemic cells derived from patients affected by chronic and juvenile myelomonocytic leukemia

RNA Pol II in global NER (XPC) deficient cells, but failed to do it in TC NER (CSB, XPD and XPG) deficient cells. Importantly, these effects were confirmed to be specific for the Rpb1 subunit of RNA Pol II, since other subunits were not affected (Rpb2 and Rpb4) as well as other factors of the transcriptional machinery, such as TBP (TFIID), p62 (TFIIH), XPD or the RPA194 subunit of the RNA Pol I. Finally, it was also demonstrated that, contrary to what occurs after DNA damage with UV light, the transcription of p53 target genes important for DNA repair, including p21 or mdm2, was irreversibly inhibited after PM01183 treatment. Together, these results show the mechanism by which PM01183 inhibits trans-activated transcription process on tumor cells.