The recent demonstration of the occurrence in rat brain and other nonpancreatic tissues of carboxypeptidase A (CPA) gene transcripts without associated catalytic activity could be ascribed to the ...presence of a soluble endogenous protein inhibitor. This tissue carboxypeptidase inhibitor (TCI), detected by the inhibition of added bovine pancreatic CPA, was purified from rat brain. Peptides were obtained by partial proteolysis of purified TCI, a protein of ≈30 kDa, and starting from their sequences, a full-length cDNA encoding a 223-amino acid protein containing three potential phosphorylation sites was cloned from a cDNA library. Its identity with TCI was shown by expression in Escherichia coli of a recombinant protein recognized by antibodies raised against native TCI and displaying characteristic CPA-inhibiting activity. TCI appears as a hardly reversible, non-competitive, and potent inhibitor of CPA1 and CPA2 (Ki≈ 3 nM) and mast-cell CPA (Ki= 16 nM), which is less potent against carboxypeptidase B (CPB; Ki= 194 nM) and inactive on various other proteases. This pattern of selectivity might be attributable to a limited homology of a 11-amino acid sequence with sequences within the activation segments of CPA and CPB known to interact with residues within their active sites. The widespread expression of TCI in a number of tissues (e.g., brain, lung, or digestive tract) and its apparently cytosolic localization point to a rather general functional role, e.g., in the control of cytosolic protein degradation.
Abstract
Histone methyl transferases (HMTs) and demethylases are chromatin modifying enzymes known to play a key role in establishing and maintaining chromatin structure and thereby contributing to ...the control of gene expression. The histone methyltransferase Enhancer of Zeste Homologue 2 (EZH2) is the catalytic component of the Polycomb Repressive Complex 2 and mediates trimethylation of lysine 27 on histone 3 (H3K27me3), which correlates with transcriptional repression. EZH2 has been widely implicated in cancer and inhibition of its catalytic activity recently emerged as a novel therapeutic approach to treat human cancers. Constellation has developed potent, selective and reversible EZH2 small molecule inhibitors that are currently being tested in clinical trials.
We have previously reported EZH2 dependencies across non-Hodgkin Lymphoma subtypes, including models harboring both wild-type and mutant EZH2. To identify other cancer types that may rely on EZH2 for survival, we carried out long term growth assays across a 200+ cancer cell line panel. We observed that over 50% of multiple myeloma cell lines show -time and -dose dependent phenotypic response to EZH2 inhibition. Similar to lymphoma, EZH2 inhibitors induce apoptosis after continuous treatment over a longer time period. To understand the underlying molecular consequences of EZH2 inhibition in multiple myeloma, we performed RNA-sequencing and ChIP-sequencing in the absence and presence of the inhibitor. We identified an EZH2-controlled transcriptional signature across various multiple myeloma models and key downstream effectors including CDKN1A in individual models.
EZH2 inhibitors such as CPI-169 achieve tumor growth inhibition in several multiple myeloma subcutaneous xenograft models at well tolerated doses, and this impact on tumor growth correlated well with target inhibition. To expand the scope of EZH2 inhibitor application in multiple myeloma, we systematically combined EZH2 inhibitors with standard of care agents, including, lenalidomide, prednisolone, bortezomib and HDAC inhibitors. We observed synergy of EZH2 inhibitors with several of these agents in vitro and in vivo and are currently exploring the molecular basis of these combinatorial effects.
In conclusion, we provide ample evidence suggesting multiple myeloma as a disease indication in which EZH2 inhibitors may show clinical benefit as a single agent and in combination with approved therapeutics.
Citation Format: Shilpi Arora, Kaylyn Williamson, Srividya Balasubramanian, Jennifer Busby, Shivani Garapaty-Rao, Charlie Hatton, Dhanalakshmi Sivanandhan, Barbara Bryant, Emmanuel Normant, Patrick Trojer. EZH2 inhibitors reveal broad EZH2 dependencies in multiple myeloma. abstract. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr PR09.
The transmembrane protein CD47 is an immunoglobulin superfamily member involved in multiple cellular processes, including cell migration, adhesion and T cell function. The interaction between CD47 ...and signal regulatory protein alpha (SIRPα), an inhibitory protein expressed on macrophages, prevents phagocytosis of CD47-expressing cells. CD47 was originally identified as a tumor antigen on human ovarian cancer and was subsequently shown to be expressed on multiple human tumor types, including both hematologic and solid tumors. In many hematologic cancers, high CD47 expression is associated with poor clinical outcomes. CD47 is also expressed at low levels on virtually all non-malignant cells, and loss of expression or changes in membrane distribution may serve as markers of aged or damaged cells, particularly on red blood cells (RBC). High expression of CD47 on tumors blocks phagocytic uptake, subsequent antigen cross-presentation and T cell activation, which collectively contribute to tumor immune evasion. Agents that block the CD47-SIRPα interaction can restore phagocytic uptake of CD47+ target cells and lower the threshold for macrophage activation, which can enhance the efficacy of therapeutic antibodies with ADCC-enabling activity. We developed and characterized a CD47 blocking antibody and evaluated its activity in multiple hematologic models.
SRF231 is a fully human monoclonal antibody that binds with high affinity to human CD47 and blocks the CD47-SIRP□ interaction. SRF231 promotes macrophage-mediated phagocytic clearance of several hematologic primary tumor samples and cell lines in vitro. For example, SRF231 increases phagocytosis of Raji tumor cell line targets with an EC50 of ~300 ng/ml. Enhanced phagocytosis is preferential for tumor cells over normal leukocytes and RBC. Tumor cell phagocytosis can be enhanced in the presence of opsonizing antibodies (e.g., anti-CD20 Ab) when co-administered with SRF231.
In vivo efficacy of SRF231 was assessed in several preclinical murine xenograft models of hematologic malignancies. Notably, SRF231 administration led to profound tumor growth inhibition in models of multiple myeloma, diffuse large B cell lymphoma and Burkitt's lymphoma as a single agent and in combination with opsonizing antibodies. In the Raji xenograft model, single agent therapy leads to 112% tumor growth inhibition. This anti-tumor activity is at least partially dependent on macrophages, as depletion of macrophages via clodronate administration leads to reduced tumor growth inhibition. Tumor-associated macrophage (TAM) numbers and polarization status are also modulated by SRF231 treatment.
In summary, the CD47 mAb SRF231 induces robust tumor cell phagocytosis and tumor clearance both alone and in combination with opsonizing antibodies in pre-clinical models of myeloma and lymphoma. These properties warrant further development of SRF231 in hematologic malignancies. SRF231 is currently in IND-enabling studies and is expected to enter clinical trials in 2017.
No relevant conflicts of interest to declare.
Treatment of gastroenteropancreatic neuroendocrine tumors (GEP-NET) is still unsatisfactory and innovative therapeutic approaches are urgently needed. Heat shock protein 90 (Hsp90) is overexpressed ...in a wide range of tumor types and is an emerging target for the treatment of cancer. However, the potential activity of Hsp90 inhibitors in GEP-NET has not yet been investigated. We studied the antineoplastic activity of the Hsp90 inhibitor IPI-504 on GEP‑NET cells, and characterized its mechanism of action. In human insulinoma (CM) and pancreatic carcinoid (BON) cells IPI-504 induced a dose-dependent growth inhibition by almost 70%. The antiproliferative effect of IPI-504 correlated with a reduction in protein levels of the IGF-1 receptor. Additionally, several proteins of the PI3K/AKT/mTOR pathway, downstream of IGF-1 receptor activation in GEP-NETs, were downregulated as a consequence of Hsp90 inhibition. Combination treatment of IPI-504 with mTOR- or AKT-inhibitors led to additive antiproliferative effects. In addition, effects of IGF-1 receptor tyrosine kinase inhibition were strongly enhanced by IPI-504. Cancer gene expression profiling and FACS analysis revealed that IPI-504 antiproliferative effects were due to both induction of cell cycle arrest and apoptosis. A modified chick chorioallantoic membrane (CAM) assay confirmed the antineoplastic activity of IPI-504 in GEP-NETs in vivo. In conclusion, this study showed that Hsp90 inhibition may be an attractive target for innovative GEP-NET treatment alone or in combination with either IGF-1R or mTOR inhibitors.
Studies indicate that pro-opiomelanocortin (POMC) is sorted to the regulated secretory pathway by binding to a sorting receptor identified as membrane-bound carboxypeptidase E (CPE) Cool et al. ...(1997) Cell 88, 73–83. The efficiency of this sorting mechanism could be enhanced if POMC molecules were to self-associate to form oligomers, prior or subsequent to binding to CPE. Using cross-linking and gel filtration techniques, we demonstrated that POMC forms oligomers at both neutral and acidic pHs and calcium was not necessary. ΔN-POMC, which lacks the N-terminal sorting signal for the regulated secretory pathway, also formed similar oligomers, indicating that the sorting and oligomerization domains are different.
Abstract
Mantle cell lymphoma (MCL) is an aggressive B lymphoid neoplasm genetically characterized by the t(11;14)(q13;q32) leading to the overexpression of cyclin D1. As a consequence of its poor ...responses to conventional chemotherapy and relatively short patient survival, new therapeutic strategies are required. Despite the promising introduction of the proteasome inhibitor bortezomib in the treatment of MCL, not all the patients respond and relapse frequently occurs. To unravel the factors involved in the acquisition of bortezomib resistance in vivo, immunodeficient mice were engrafted with a set of MCL cell lines with different levels of sensitivity to the drug, followed by gene expression profiling of the tumors and functional validation of the identified gene signatures. We observed an increased tumorigenicity of bortezomib-resistant MCL cells in vivo, that was associated with plasmacytic differentiation features, like IRF4 and Blimp-1 upregulation. As the immunomoduladory drug lenalidomide has been shown to modulate IRF4 expression in various B-cell malignancies, we assessed its activity in in vitro and in vivo settings by means of flow cytometry, western blot, antibody array, real-time PCR, immunofluorescence, in vivo imaging, and immunohistochemistry. In vitro, lenalidomide as single agent was found to exert antitumor activity in 4/11 MCL cell lines, corresponding to those cells with either primary or acquired resistance to bortezomib. Lenalidomide-treated cells showed decreased IRF4 expression, increased cytosolic amounts of p27 and caspase-dependent apoptosis. Accordingly, mice bearing bortezomib-resistant tumors and treated for 3 weeks with a lenalidomide regimen of 10-50 mg/kg/day, showed a 30 to 45% reduction in tumor burden when compared to vehicle-treated mice (p=0.04), with several hallmarks of lenalidomide activity, like downregulation of IRF4 and its target gene MYC, decreased mitotic index, p27 cytosolic accumulation and caspase-3 processing. Importantly, the inhibition of tumor growth induced by the combination of lenalidomide with bortezomib (0.15 mg/kg, twice a week) was 37% and 66% greater than that for lenalidomide alone and vehicle arms, respectively (p=0.02). Moreover, repression of MYC in bortezomib-resistant cells by gene knockdown or treatment with CPI203, a BET bromodomain inhibitor, synergistically induced cell death when combined with lenalidomide therapy. Accordingly, co-treatment of mice with lenalidomide plus CPI203 synergistically reduced MYC and IRF4 expression and tumor burden, and induced caspase processing. Together, these results suggest that exacerbated IRF4/MYC signaling is associated to bortezomib resistance in MCL in vivo and warrant clinical evaluation of lenalidomide plus BET inhibitor combination in MCL cases refractory to proteasome inhibition.
Citation Format: Alexandra Moros, Vanina Rodriguez, Ifigenia Saborit-Villarroya, Arnau Montraveta, Patricia Balsas, Peter Sandy, Antonio Martinez, Emmanuel Normant, Patricia Perez-Galan, Elias Campo, Dolors Colomer, Gael Roue. Synergistic anti-tumor activity of lenalidomide with the BET bromodomain inhibitor CPI203 in bortezomib-resistant mantle cell lymphoma. abstract. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1691. doi:10.1158/1538-7445.AM2014-1691
Several Hsp90 (heat shock protein 90) inhibitors are currently under clinical evaluation as anticancer agents. However, the correlation between the duration and magnitude of Hsp90 inhibition and the ...downstream effects on client protein degradation and cancer cell growth inhibition has not been thoroughly investigated. To investigate the relationship between Hsp90 inhibition and cellular effects, we developed a method that measures drug occupancy on Hsp90 after treatment with the Hsp90 inhibitor IPI-504 in living cells and in tumor xenografts. In cells, we find the level of Hsp90 occupancy to be directly correlated with cell growth inhibition. At the molecular level, the relationship between Hsp90 occupancy and Hsp90 client protein degradation was examined for different client proteins. For sensitive Hsp90 clients (e.g. HER2 (human epidermal growth factor receptor 2), client protein levels directly mirror Hsp90 occupancy at all time points after IPI-504 administration. For insensitive client proteins, we find that protein abundance matches Hsp90 occupancy only after prolonged incubation with drug. Additionally, we investigate the correlation between plasma pharmacokinetics (PK), tumor PK, pharmacodynamics (PD) (client protein degradation), tumor growth inhibition, and Hsp90 occupancy in a xenograft model of human cancer. Our results indicate Hsp90 occupancy to be a better predictor of PD than either plasma PK or tumor PK. In the nonsmall cell lung cancer xenograft model studied, a linear correlation between Hsp90 occupancy and tumor growth inhibition was found. This novel binding assay was evaluated both in vitro and in vivo and could be used as a pharmacodynamic readout in the clinic.
Previous studies have shown that the prohormone POMC is sorted to the
regulated secretory pathway (RSP), at the trans-Golgi
network, by binding of a conformation-dependent sorting signal to a
sorting ...receptor, identified as membrane-bound carboxypeptidase E (CPE)
(Cool et al., 1997, Cell, 88:73–83). In
this study, the role of CPE as a sorting receptor for other RSP
proteins that contain sorting signals (proinsulin, proenkephalin, and
chromogranin A) was investigated in neuroendocrine cells (Neuro-2a)
stably expressing CPE antisense RNA. Whereas these cells were depleted
of CPE by greater than 85%, electron microscopy showed that they
contain dense core secretory granules identical to wild-type Neuro-2a
cells, indicating that CPE is not essential for granulogenesis.
Secretion and immunocytochemical studies showed that, in wild-type
Neuro-2a cells, endogenous proenkephalin and transfected
proinsulin/insulin were localized to punctate secretory granules and
were released via the RSP. However, in CPE-depleted cells, these two
prohormones were released constitutively and had a Golgi-like
distribution but were not localized to punctate secretory granules. In
contrast, chromogranin A was present in punctate secretory granules and
released via the RSP, in wild-type and CPE-depleted Neuro-2a cells.
Thus, the sorting of proinsulin and proenkephalin to the RSP, like
POMC, necessitates binding to CPE, and hence, CPE acts as a common
sorting receptor for targeting these prohormones to the RSP. In
contrast, the sorting signal of chromogranin A does not use CPE as a
sorting receptor, suggesting the existence of other sorting receptors
for the RSP.
The updated affiliation list is shown below and has been updated in the article. 1Istituto Nazionale Tumori IRCCS Fondazione 'G. Pascale', Naples, Italy 2Earle A. Chiles Research Institute, ...Providence Cancer Institute, Portland, Oregon, USA 3Harvard Medical School, Boston, Massachusetts, USA 4Georgetown Lombardi Comprehensive Cancer Center, Washington DC, USA 5University of Wisconsin Clinical Cancer Center, Madison, Wisconsin, USA 6Johns Hopkins University School of Medicine, Sidney Kimmel Comprehensive Cancer Center, Baltimore, Maryland, USA 7Research, Parker Institute for Cancer Immunotherapy, San Francisco, California, USA 8ESSA Pharma Inc, Redwood City, California, USA 9IGM Biosciences Inc, Mountain View, California, USA 10Medical Oncology - Amsterdam University Medical Centers, Vrije Universiteit-Cancer Center Amsterdam, Amsterdam, The Netherlands 11AIVITA Biomedical, Inc, Irvine, California, USA 12Herbert Irving Comprehensive Cancer Center, Columbia University Medical Center, New York, New York, USA 13UPMC Hillman Cancer Center, Pittsburgh, Pennsylvania, USA 14Pathology and Medicine, Immunology and Cancer Program, University of Chicago, Chicago, Illinois, USA 15National Cancer Institute, Bethesda, Maryland, USA 16Dana Farber Cancer Institute, Boston, Massachusetts, USA 17University of Texas MD Anderson Cancer Center, Houston, Texas, USA 18Bill & Melinda Gates Medical Research Institute, Cambridge, Massachusetts, USA 19Immuneering Corp New York, New York, New York, USA 20University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA 21Carbone Cancer Center, University of Wisconsin-Madison, Madison, Wisconsin, USA 22Medical Oncology, City of Hope National Medical Center, Duarte, California, USA 23Refuge Biotechnologies, Menlo Park, California, USA 24Thomas Jefferson Medical College, Philadelphia, Pennsylvania, USA 25Massachusetts General Hospital Cancer Center, Harvard Medical School, Massachusetts General Hospital, Boston, Massachusetts, USA 26ESSA Pharma Inc, Palo Alto, California, USA 27Oncology, University of Lausanne, Lausanne, VD, Switzerland 28Pediatrics, University of Wisconsin Madison, Madison, Wisconsin, USA 29Massachusetts Institute of Technology Koch Institute for Integrative Cancer Research, Cambridge, Massachusetts, USA 30Yale Cancer Center, Yale School of Medicine, New Haven, Connecticut, USA 31Holden Comprehensive Cancer Center, The University of Iowa, Iowa City, Iowa, USA 32MacroGenics Inc, Rockville, Maryland, USA 33Laura and Isaac Perlmutter Comprehensive Cancer Center, NYU Langone Medical Center, New York, New York, USA 3) In the main text, The sentence ‘The hypoxia and profound inflammatory response associated with the pneumonitis observed with the severe acute respiratory virus coronavirus-2 SARS-COV-2 virus…’ now reads ‘The hypoxia and profound inflammatory response associated with the pneumonitis observed with the SARS-CoV-2 virus…’ The sentence ‘…including tocilizumab and sarilumab for use on a compassionate basis to critically ill hospitalized COVID-19-infected patients during this extraordinary situation’ now reads ‘…including tocilizumab and sarilumab for use on a compassionate basis to critically ill hospitalized SARS-CoV-2-infected patients during this extraordinary situation’ 4) To acknowledge medical writing support, the acknowledgment section has been updated to read: ‘The authors thank the clinicians working tirelessly on the frontlines of the COVID-19 pandemic. The authors also acknowledge SITC staff for their contributions including Sam Million Weaver, PhD for medical writing and editorial support and Angela Kilbert for project management and assistance.
PDF
