LINGO3 regulates mucosal tissue regeneration and promotes TFF2 dependent recovery from colitis.

Aim: Recovery of damaged mucosal surfaces following inflammatory insult requires diverse regenerative mechanisms that remain poorly defined. Previously, we demonstrated that the reparative actions of Trefoil Factor 3 (TFF3) depend upon the enigmatic receptor, leucine rich repeat and immunoglobulin-like domain containing nogo receptor 2 (LINGO2). This study examined the related orphan receptor LINGO3 in the context of intestinal tissue damage to determine whether LINGO family members are generally important for mucosal wound healing and maintenance of the intestinal stem cell (ISC) compartment needed for turnover of mucosal epithelium.Methods and Results: We find that LINGO3 is broadly expressed on human enterocytes and sparsely on discrete cells within the crypt niche, that contains ISCs. Loss of function studies indicate that LINGO3 is involved in recovery of normal intestinal architecture following dextran sodium sulfate (DSS)-induced colitis, and that LINGO3 is needed for therapeutic action of the long acting TFF2 fusion protein (TFF2-Fc), including a number of signaling pathways critical for cell proliferation and wound repair. LINGO3-TFF2 protein-protein interactions were relatively weak however and LINGO3 was only partially responsible for TFF2 induced MAPK signaling suggesting additional un-identified components of a receptor complex. However, deficiency in either TFF2 or LINGO3 abrogated budding/growth of intestinal organoids and reduced expression of the intestinal ISC gene leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5), indicating homologous roles for these proteins in tissue regeneration, possibly via regulation of ISCs in the crypt niche.Conclusion: We propose that LINGO3 serves a previously unappreciated role in promoting mucosal wound healing.

NPM-ALK-Induced Reprogramming of Mature TCR-Stimulated T Cells Results in Dedifferentiation and Malignant Transformation.

Fusion genes including NPM-ALK can promote T-cell transformation, but the signals required to drive a healthy T cell to become malignant remain undefined. In this study, we introduce NPM-ALK into primary human T cells and demonstrate induction of the epithelial-to-mesenchymal transition (EMT) program, attenuation of most T-cell effector programs, reemergence of an immature epigenomic profile, and dynamic regulation of c-Myc, E2F, and PI3K/mTOR signaling pathways early during transformation. A mutant of NPM-ALK failed to bind several signaling complexes including GRB2/SOS, SHC1, SHC4, and UBASH3B and was unable to transform T cells. Finally, T-cell receptor (TCR)-generated signals were required to achieve T-cell transformation, explaining how healthy individuals can harbor T cells with NPM-ALK translocations. These findings describe the fundamental mechanisms of NPM-ALK-mediated oncogenesis and may serve as a model to better understand factors that regulate tumor formation. SIGNIFICANCE: This investigation into malignant transformation of T cells uncovers a requirement for TCR triggering, elucidates integral signaling complexes nucleated by NPM-ALK, and delineates dynamic transcriptional changes as a T cell transforms.See related commentary by Spasevska and Myklebust, p. 3160.

Cellular context of IL-33 expression dictates impact on anti-helminth immunity.

Interleukin-33 (IL-33) is a pleiotropic cytokine that can promote type 2 inflammation but also drives immunoregulation through Foxp3+Treg expansion. How IL-33 is exported from cells to serve this dual role in immunosuppression and inflammation remains unclear. Here, we demonstrate that the biological consequences of IL-33 activity are dictated by its cellular source. Whereas IL-33 derived from epithelial cells stimulates group 2 innate lymphoid cell (ILC2)-driven type 2 immunity and parasite clearance, we report that IL-33 derived from myeloid antigen-presenting cells (APCs) suppresses host-protective inflammatory responses. Conditional deletion of IL-33 in CD11c-expressing cells resulted in lowered numbers of intestinal Foxp3+Treg cells that express the transcription factor GATA3 and the IL-33 receptor ST2, causing elevated IL-5 and IL-13 production and accelerated anti-helminth immunity. We demonstrate that cell-intrinsic IL-33 promoted mouse dendritic cells (DCs) to express the pore-forming protein perforin-2, which may function as a conduit on the plasma membrane facilitating IL-33 export. Lack of perforin-2 in DCs blocked the proliferative expansion of the ST2+Foxp3+Treg subset. We propose that perforin-2 can provide a plasma membrane conduit in DCs that promotes the export of IL-33, contributing to mucosal immunoregulation under steady-state and infectious conditions.

ATM inhibition overcomes resistance to histone deacetylase inhibitor due to p21 induction and cell cycle arrest.

The antiproliferative effect induced by histone deactylase inhibitors (HDACi) is associated with the up-regulated expression of the cyclin-dependent kinase inhibitor p21. Paradoxically, the increased expression of p21 correlates with a reduced cell killing to the drug. The direct targeting of p21 is not feasible. An alternate approach could selectively target factors upstream or downstream of p21 that affect one or more specific aspects of p21 function. HDAC inhibitors appear to activate p21 expression via ataxia telangiectasia mutated (ATM) activity. KU60019, a specific ATM inhibitor, has shown to decrease the p21 protein levels in a concentration dependent manner. We explored the potential synergistic interaction of the ATM inhibitor with romidepsin, given the potential complementary impact around p21. A synergistic cytotoxic effect was observed in all lymphoma cell lines examined when the HDACi was combined with KU60019. The increase in apoptosis correlates with decreased expression of p21 due to the ATM inhibitor. KU60019 decreased expression of the cyclin-dependent kinase inhibitor at the transcriptional level, compromising the ability of HDACi to induce p21 and cell cycle arrest and ultimately facilitating a shift toward the apoptotic phase. Central to the increased apoptosis observed when romidepsin is combined with KU60019 is the reduced expression of p21 and the absence of a G2/M cell cycle arrest that would be exploited by the tumor cells to evade the cytotoxic effect of the HDAC inhibitor. We believe this strategy may offer a promising way to identify rational combinations for HDACi directed therapy, improving their activity in malignant disease.

TFF3 interacts with LINGO2 to regulate EGFR activation for protection against colitis and gastrointestinal helminths.

Intestinal epithelial cells (IEC) have important functions in nutrient absorption, barrier integrity, regeneration, pathogen-sensing, and mucus secretion. Goblet cells are a specialized cell type of IEC that secrete Trefoil factor 3 (TFF3) to regulate mucus viscosity and wound healing, but whether TFF3-responsiveness requires a receptor is unclear. Here, we show that leucine rich repeat receptor and nogo-interacting protein 2 (LINGO2) is essential for TFF3-mediated functions. LINGO2 immunoprecipitates with TFF3, co-localizes with TFF3 on the cell membrane of IEC, and allows TFF3 to block apoptosis. We further show that TFF3-LINGO2 interactions disrupt EGFR-LINGO2 complexes resulting in enhanced EGFR signaling. Excessive basal EGFR activation in Lingo2 deficient mice increases disease severity during colitis and augments immunity against helminth infection. Conversely, TFF3 deficiency reduces helminth immunity. Thus, TFF3-LINGO2 interactions de-repress inhibitory LINGO2-EGFR complexes, allowing TFF3 to drive wound healing and immunity.

Group 2 Innate Lymphoid Cells (ILC2): Type 2 Immunity and Helminth Immunity.

Group 2 innate lymphoid cells (ILC2) have emerged as a major component of type 2 inflammation in mice and humans. ILC2 secrete large amounts of interleukins 5 and 13, which are largely responsible for host protective immunity against helminth parasites because these cytokines induce profound changes in host physiology that include: goblet cell metaplasia, mucus accumulation, smooth muscle hypercontractility, eosinophil and mast cell recruitment, and alternative macrophage activation (M2). This review covers the initial recognition of ILC2 as a distinct cell lineage, the key studies that established their biological importance, particularly in helminth infection, and the new directions that are likely to be the focus of emerging work that further explores this unique cell population in the context of health and disease.

Mechanisms of Acquired Drug Resistance to the HDAC6 Selective Inhibitor Ricolinostat Reveals Rational Drug-Drug Combination with Ibrutinib.

Purpose: Pan-class I/II histone deacetylase (HDAC) inhibitors are effective treatments for select lymphomas. Isoform-selective HDAC inhibitors are emerging as potentially more targeted agents. ACY-1215 (ricolinostat) is a first-in-class selective HDAC6 inhibitor. To better understand the discrete function of HDAC6 and its role in lymphoma, we developed a lymphoma cell line resistant to ACY-1215.Experimental Design: The diffuse large B-cell lymphoma cell line OCI-Ly10 was exposed to increasing concentrations of ACY-1215 over an extended period of time, leading to the development of a resistant cell line. Gene expression profiling (GEP) was performed to investigate differentially expressed genes. Combination studies of ACY-1215 and ibrutinib were performed in cell lines, primary human lymphoma tissue, and a xenograft mouse model.Results: Systematic incremental increases in drug exposure led to the development of distinct resistant cell lines with IC50 values 10- to 20-fold greater than that for parental lines. GEP revealed upregulation of MAPK10, HELIOS, HDAC9, and FYN, as well as downregulation of SH3BP5 and LCK. Gene-set enrichment analysis (GSEA) revealed modulation of the BTK pathway. Ibrutinib was found to be synergistic with ACY-1215 in cell lines as well as in 3 primary patient samples of lymphoma. In vivo confirmation of antitumor synergy was demonstrated with a xenograft of DLBCL.Conclusions: The development of this ACY-1215-resistant cell line has provided valuable insights into the mechanistic role of HDAC6 in lymphoma and offered a novel method to identify rational synergistic drug combinations. Translation of these findings to the clinic is underway. Clin Cancer Res; 23(12); 3084-96. ©2016 AACR.

The combination of hypomethylating agents and histone deacetylase inhibitors produce marked synergy in preclinical models of T-cell lymphoma.

T-cell lymphomas (TCL) are aggressive lymphomas usually treated with CHOP (cyclophsophamide, doxorubicin, vincristine, prednisolone)-like regimens upfront. Recent data suggest that TCL are driven by epigenetic defects, potentially rendering them sensitive to epigenetic therapies. We explored the therapeutic merits of a combined epigenetic platform using histone deacetylase inhibitors (HDACIs) and DNA methyltransferase inhibitors (DNMT) in in vitro and in vivo models of TCL. The 50% inhibitory concentration (IC50 ) values revealed romidepsin was the most potent HDACI, with an IC50 in the low nanomolar range. The combination with a hypomethylating agent produced synergy across all cell lines, which was confirmed in cytotoxicity and apoptosis assays. An in vivo xenograft study demonstrated inhibition of tumour growth in the combination cohort compared to the single agent. Gene expression array and global methylation profiling revealed differentially expressed genes and modulated pathways for each of the single treatment conditions and the combination. Most of the effects induced by the single agent treatment were maintained in the combination group. In total, 944 unique genes were modulated by the combination treatment, supporting the hypothesis of molecular synergism. These data suggest combinations of hypomethylating agents and HDACIs are synergistic in models of TCL, which is supported at the molecular level.

Dual Targeting of Protein Degradation Pathways with the Selective HDAC6 Inhibitor ACY-1215 and Bortezomib Is Synergistic in Lymphoma.

PURPOSE: Pan-class histone deacetylase (HDAC) inhibitors are effective treatments for select lymphomas. Isoform-selective HDAC inhibitors are emerging as potentially more targeted agents. HDAC6 is a class IIb deacetylase that facilitates misfolded protein transport to the aggresome for degradation. We investigated the mechanism and therapeutic impact of the selective HDAC6 inhibitor ACY-1215 alone and in combination with bortezomib in preclinical models of lymphoma. EXPERIMENTAL DESIGN: Concentration-effect relationships were defined for ACY-1215 across 16 lymphoma cell lines and for synergy with bortezomib. Mechanism was interrogated by immunoblot and flow cytometry. An in vivo xenograft model of DLBCL was used to confirm in vitro findings. A collection of primary lymphoma samples were surveyed for markers of the unfolded protein response (UPR). RESULTS: Concentration-effect relationships defined maximal cytotoxicity at 48 hours with IC50 values ranging from 0.9 to 4.7 µmol/L. Strong synergy was observed in combination with bortezomib. Treatment with ACY-1215 led to inhibition of the aggresome evidenced by acetylated α-tubulin and accumulated polyubiquitinated proteins and upregulation of the UPR. All pharmacodynamic effects were enhanced with the addition of bortezomib. Findings were validated in vivo where mice treated with the combination demonstrated significant tumor growth delay and prolonged overall survival. Evaluation of a collection of primary lymphoma samples for markers of the UPR revealed increased HDAC6, GRP78, and XBP-1 expression as compared with reactive lymphoid tissue. CONCLUSIONS: These data are the first results to demonstrate that dual targeting of protein degradation pathways represents an innovative and rational approach for the treatment of lymphoma.

Aurora A Kinase Inhibition Selectively Synergizes with Histone Deacetylase Inhibitor through Cytokinesis Failure in T-cell Lymphoma.

PURPOSE: Aurora A kinase (AAK) is expressed exclusively during mitosis, and plays a critical role in centrosome duplication and spindle formation. Alisertib is a highly selective AAK inhibitor that has demonstrated marked clinical activity of alisertib across a spectrum of lymphomas, though particularly in patients with T-cell lymphoma (TCL). We sought to compare and contrast the activity of alisertib in preclinical models of B-cell lymphoma (BCL) and TCL, and identify combinations worthy of clinical study. High-throughput screening of pralatrexate, the proteasome inhibitor (ixazomib), and the histone deacetylase (HDAC) inhibitor (romidepsin) revealed that only romidepsin synergized with alisertib, and only in models of TCL. We discovered that the mechanism of synergy between AAK inhibitors and HDAC inhibitors appears to be mediated through cytokinesis failure. EXPERIMENTAL DESIGN: A high-throughput screening approach was used to identify drugs that were potentially synergistic in combination with alisertib. Live-cell imaging was used to explore the mechanistic basis for the drug: drug interaction between alisertib and romidepsin. An in vivo xenograft TCL model was used to confirm in vitro results. RESULTS: In vitro, alisertib exhibited concentration-dependent cytotoxicity in BCL and TCL cell lines. Alisertib was synergistic with romidepsin in a T-cell-specific fashion that was confirmed in vivo. Live-cell imaging demonstrated that the combination treatment resulted in profound cytokinesis failure. CONCLUSIONS: These data strongly suggest that the combination of alisertib and romidepsin is highly synergistic in TCL through modulation of cytokinesis and merits clinical development.

The novel kinesin spindle protein (KSP) inhibitor SB-743921 exhibits marked activity in in vivo and in vitro models of aggressive large B-cell lymphoma.

The kinesin spindle protein (KSP) is a mitotic protein essential for cell cycle control and motility. SB-743921 (hereafter SB-921) is an inhibitor that selectively targets the ATP-binding domain of the KSP. The preclinical activity of SB-921 was evaluated in models of diffuse large B-cell lymphoma (DLBCL). The cytotoxicity of SB-921 was evaluated in a series of germinal center (GC-DLBCL) and post-germinal center (ABC-DLBCL) DLBCL cell lines and a murine lymphoma xenograft model. GC-DLBCL lines generally demonstrated greater sensitivity to SB-921. IC50 values ranged between 1 nM and 900 nM for GC-DLBCL compared to 1 nM to 10 µM for ABC lines. SB-921 demonstrated marked activity in a xenograft model of Ly-1 (GC-DLBCL). While SB-921 was relatively more active in GC derived cell lines, ABC-derived lines still underwent apoptosis at higher concentrations. These results demonstrate that SB-921 inhibits proliferation and induces apoptosis in both GC-DLBCL and ABC-DLBCL.

The addition of granulocyte-colony stimulating factor shifts the dose limiting toxicity and markedly increases the maximum tolerated dose and activity of the kinesin spindle protein inhibitor SB-743921 in patients with relapsed or refractory lymphoma: results of an international, multicenter phase I/II study.

This was a phase I study of SB-743921 (SB-921) in patients with relapsed/refractory lymphoma. Previous studies established that neutropenia was the only dose limiting toxicity (DLT). The primary objective was to determine the DLT, maximum tolerated dose (MTD) and efficacy of SB-921 with and without granulocyte-colony stimulating factor (G-CSF). Sixty-eight patients were enrolled, 42 without G-CSF, 26 with G-CSF. In the cohort without G-CSF, SB-921 doses ranged from 2 to 7 mg/m(2), with 6 mg/m(2) being the MTD. In the cohort with G-CSF support, doses of 6-10 mg/m(2) were administered, with 9 mg/m(2) being the MTD, representing a 50% increase in dose density. Fifty-six patients were evaluable for efficacy. Four of 55 patients experienced a partial response (three in Hodgkin lymphoma and one in non-Hodgkin lymphoma, all at doses ≥ 6 mg/m(2)); 19 patients experienced stable disease, 33 patients developed progression of disease. G-CSF shifted the DLT from neutropenia to thrombocytopenia, allowing for a 50% increase in dose density. Responses were seen at higher doses with G-CSF support.

Preclinical pharmacologic evaluation of pralatrexate and romidepsin confirms potent synergy of the combination in a murine model of human T-cell lymphoma.

PURPOSE: T-cell lymphomas (TCL) are aggressive diseases, which carry a poor prognosis. The emergence of new drugs for TCL has created a need to survey these agents in a rapid and reproducible fashion, to prioritize combinations which should be prioritized for clinical study. Mouse models of TCL that can be used for screening novel agents and their combinations are lacking. Developments in noninvasive imaging modalities, such as surface bioluminescence (SBL) and three-dimensional ultrasound (3D-US), are challenging conventional approaches in xenograft modeling relying on caliper measurements. The recent approval of pralatrexate and romidepsin creates an obvious combination that could produce meaningful activity in TCL, which is yet to be studied in combination. EXPERIMENTAL DESIGN: High-throughput screening and multimodality imaging approach of SBL and 3D-US in a xenograft NOG mouse model of TCL were used to explore the in vitro and in vivo activity of pralatrexate and romidepsin in combination. Corresponding mass spectrometry-based pharmacokinetic and immunohistochemistry-based pharmacodynamic analyses of xenograft tumors were performed to better understand a mechanistic basis for the drug:drug interaction. RESULTS: In vitro, pralatrexate and romidepsin exhibited concentration-dependent synergism in combination against a panel of TCL cell lines. In a NOG murine model of TCL, the combination of pralatrexate and romidepsin exhibited enhanced efficacy compared with either drug alone across a spectrum of tumors using complementary imaging modalities, such as SBL and 3D-US. CONCLUSIONS: Collectively, these data strongly suggest that the combination of pralatrexate and romidepsin merits clinical study in patients with TCLs.

Pralatrexate pharmacology and clinical development.

Folates are well known to be essential for many cellular processes, including cellular proliferation. As a consequence, antifolates, the fraudulent mimics of folic acid, have been shown to be potent therapeutic agents in many cancers. Over the past several decades, efforts to improve on this class of drugs have met with little success. Recently, one analog specifically designed to have high affinity for the reduced folate carrier, which efficiently internalizes natural folates and antifolates, has been shown to be very active in T-cell lymphoma. Pralatrexate, approved by the U.S. Food and Drug Administration in 2009, is highly active across many lymphoid malignancies, including chemotherapy-resistant T-cell lymphoma. Emerging combination studies have now shown that pralatrexate is highly synergistic with gemcitabine, histone deacetylase inhibitors like romidepsin and bortezomib. These insights are leading to a number of novel phase I and II combination studies which could challenge existing regimens like CHOP, and improve the outcome of patients with T-cell lymphoma.

Murine models in mantle cell lymphoma.

Mantle cell lymphoma (MCL), an aggressive, heterogeneous B-cell lymphoma associated with a relatively short survival has been challenging to study in the laboratory due to the lack of in vitro and in vivo models that accurately recapitulate the disease. Advancement has been made in the characterization of MCL cell lines through the generation of the ATCC MCL bank, enabling their use in xenograft murine models. These models provide valuable but limited information for the preclinical evaluation and development of targeted therapies for MCL despite their deficiencies of a functioning immune system and correct micro-environment. Currently, there is only one double transgenic murine model known to develop spontaneous MCL. There is an urgency to develop innovative transgenic murine models that could be used to better predict therapeutic responses and precisely decipher mechanisms of action, to foster refinement of novel therapeutics for mantle cell lymphoma.