Combined inhibition of p97 and the proteasome causes lethal disruption of the secretory apparatus in multiple myeloma cells.

Inhibition of the proteasome is a widely used strategy for treating multiple myeloma that takes advantage of the heavy secretory load that multiple myeloma cells (MMCs) have to deal with. Resistance of MMCs to proteasome inhibition has been linked to incomplete disruption of proteasomal endoplasmic-reticulum (ER)-associated degradation (ERAD) and activation of non-proteasomal protein degradation pathways. The ATPase p97 (VCP/Cdc48) has key roles in mediating both ERAD and non-proteasomal protein degradation and can be targeted pharmacologically by small molecule inhibition. In this study, we compared the effects of p97 inhibition with Eeyarestatin 1 and DBeQ on the secretory apparatus of MMCs with the effects induced by the proteasome inhibitor bortezomib, and the effects caused by combined inhibition of p97 and the proteasome. We found that p97 inhibition elicits cellular responses that are different from those induced by proteasome inhibition, and that the responses differ considerably between MMC lines. Moreover, we found that dual inhibition of both p97 and the proteasome terminally disrupts ER configuration and intracellular protein metabolism in MMCs. Dual inhibition of p97 and the proteasome induced high levels of apoptosis in all of the MMC lines that we analysed, including bortezomib-adapted AMO-1 cells, and was also effective in killing primary MMCs. Only minor toxicity was observed in untransformed and non-secretory cells. Our observations highlight non-redundant roles of p97 and the proteasome in maintaining secretory homeostasis in MMCs and provide a preclinical conceptual framework for dual targeting of p97 and the proteasome as a potential new therapeutic strategy in multiple myeloma

Poly(ADP-ribose) polymerase family member 14 (PARP14) is a novel effector of the JNK2-dependent pro-survival signal in multiple myeloma

Copyright @ 2013 Macmillan Publishers Limited. This is the author's accepted manuscript. The final published article is available from the link below.Regulation of cell survival is a key part of the pathogenesis of multiple myeloma (MM). Jun N-terminal kinase (JNK) signaling has been implicated in MM pathogenesis, but its function is unclear. To elucidate the role of JNK in MM, we evaluated the specific functions of the two major JNK proteins, JNK1 and JNK2. We show here that JNK2 is constitutively activated in a panel of MM cell lines and primary tumors. Using loss-of-function studies, we demonstrate that JNK2 is required for the survival of myeloma cells and constitutively suppresses JNK1-mediated apoptosis by affecting expression of poly(ADP-ribose) polymerase (PARP)14, a key regulator of B-cell survival. Strikingly, we found that PARP14 is highly expressed in myeloma plasma cells and associated with disease progression and poor survival. Overexpression of PARP14 completely rescued myeloma cells from apoptosis induced by JNK2 knockdown, indicating that PARP14 is critically involved in JNK2-dependent survival. Mechanistically, PARP14 was found to promote the survival of myeloma cells by binding and inhibiting JNK1. Moreover, inhibition of PARP14 enhances the sensitization of MM cells to anti-myeloma agents. Our findings reveal a novel regulatory pathway in myeloma cells through which JNK2 signals cell survival via PARP14, and identify PARP14 as a potential therapeutic target in myeloma.Kay Kendall Leukemia Fund, NIH, Cancer Research UK, Italian Association for Cancer Research and the Foundation for Liver Research

CD4+ T Cell-Derived IL-2 Signals during Early Priming Advances Primary CD8+ T Cell Responses

Stimulating naïve CD8+ T cells with specific antigens and costimulatory signals is insufficient to induce optimal clonal expansion and effector functions. In this study, we show that the activation and differentiation of CD8+ T cells require IL-2 provided by activated CD4+ T cells at the initial priming stage within 0–2.5 hours after stimulation. This critical IL-2 signal from CD4+ cells is mediated through the IL-2Rβγ of CD8+ cells, which is independent of IL-2Rα. The activation of IL-2 signaling advances the restriction point of the cell cycle, and thereby expedites the entry of antigen-stimulated CD8+ T-cell into the S phase. Besides promoting cell proliferation, IL-2 stimulation increases the amount of IFNγ and granzyme B produced by CD8+ T cells. Furthermore, IL-2 at priming enhances the ability of P14 effector cells generated by antigen activation to eradicate B16.gp33 tumors in vivo. Therefore, our studies demonstrate that a full CD8+ T-cell response is elicited by a critical temporal function of IL-2 released from CD4+ T cells, providing mechanistic insights into the regulation of CD8+ T cell activation and differentiation

The distinct role of CD4+ and CD8+ T-cells during the anti-tumour effects of targeted superantigens

To target T-cells to the tumour area we created a recombinant protein of the bacterial superantigen (SAg) Staphylococcal enterotoxin A (SEA) and the Fab-fragment of a tumour-reactive antibody. This antibody-targeted SAg immunotherapy therapy has been shown to be highly efficient, eliminating > 95% of the pulmonary metastasis in mice carrying established melanoma micrometastases. Earlier studies demonstrated that elimination of the C215-expressing B16-melanoma lung metastasis was dependent on interferon (IFN)-γ release and expression of perforin. In the present study, therapeutic effector functions were analysed both locally at the tumour site and systemically in the spleen. In order to elucidate the role of each T-cell subset during Fab–SEA therapy, CD4 knock-out (KO) and CD8 KO mice were used. Tumour size reduction was statistically significant in Fab–SEA-based tumour therapy in both types of T-cell-deficient mice compared to wild-type mice. CD4 KO mice displayed a drastic reduction in the number of tumour-infiltrating macrophages and CD8+ T-cells. Therapy-induced accumulation of perforin-containing cells at the tumour site was significantly impaired in CD8 KO mice, and marginally in CD4 KO mice. Moreover, CD4 KO mice failed to produce substantial amounts of the tumour suppressive cytokine IFN-γ. This is in sharp contrast to normal mice where a massive local release was recorded. CD8 KO mice displayed a spontaneous production of interleukin (IL)-4 and IL-10 locally in the tumour. Neither normal nor CD4 KO mice produced detectable levels of these Th-2-associated cytokines. The high level of IL-10 was demonstrated to inhibit Fab–SEA tumour therapy, since the therapeutic efficacy was significantly higher in IL-10 KO mice. These results illustrate the importance of a finely tuned cellular collaboration to regulate the various phases of an efficient anti-tumour immune response. © 1999 Cancer Research Campaig

Impact of route and adequacy of nutritional intake on outcomes of allogeneic haematopoietic cell transplantation for haematologic malignancies.

BACKGROUND: Allogeneic haematopoietic cell transplantation (HCT) is often associated with poor oral intake due to painful mucositis and gastrointestinal sequalae that occur following a preparative regimen of intensive chemotherapy and/or total body radiation. Although attractive to assume that optimal nutrition improves HCT outcomes, there are limited data to support this. It is also unclear whether artificial nutrition support should be provided as enteral tube feeding or parenteral nutrition (PN). METHODS: We analysed day-100 non-relapse mortality (NRM), incidence of acute graft-versus-host disease (GvHD), acute gastrointestinal GvHD, 5-year survival and GvHD-free/relapse-free survival (GRFS) according to both route and adequacy of nutritional intake prior to neutrophil engraftment, together with other known prognostic factors, in a retrospective cohort of 484 patients who underwent allogeneic HCT for haematologic malignancy between 2000 and 2014. RESULTS: Multivariate analyses showed increased NRM with inadequate nutrition (hazard ratio (HR) 4.1; 95% confidence interval (CI) 2.2-7.2) and adequate PN (HR 2.9; 95% CI 1.6-5.4) compared to adequate enteral nutrition (EN) both P < .001. There were increased incidences of gastrointestinal GvHD of any stage and all GvHD ≥ grade 2 in patients who received PN (odds ratio (OR) 2.0; 95% CI 1.2-3.3; P = .006, and OR 1.8; 95% CI 1.1-3.0; P = .018, respectively), compared to adequate EN. Patients who received adequate PN and inadequate nutrition also had reduced probabilities of survival and GRFS at 5 years. CONCLUSION: Adequate EN during the early transplantation course is associated with reduced NRM, improved survival and GRFS at 5 years. Furthermore, adequate EN is associated with lower incidence of overall and gut acute GvHD than PN, perhaps because of its ability to maintain mucosal integrity, modulate the immune response to intensive chemo/radiotherapy and support the gastrointestinal tract environment, including gut microflora

Development of cation-directed acylation reactions

This thesis describes work conducted towards the enantioselective acylation of ketones using phase-transfer catalysis. Chapter 1 introduces the field of ketone acylation, with a focus on catalytic methods accompanied by the generation of axes, planes or points of chirality during these transformations. Chapters 2 describes the synthesis and evaluation of a variety of different frameworks for enantioselective ketone O-acylation; we highlight the selectivity and reactivity challenges faced when attempting this transformation (Scheme I). Chapter 3 focuses on the intramolecular O-acylation of enolates to generate axially chiral lactones. The development of these systems led to the discovery that the C/O-selectivity of this reaction was dependent on the steric hindrance about the axis of rotation, and C-acylation could be favoured under the correct conditions. Chapter 4 describes the conception and development of an enantioselective C-acylation reaction, culminating in the first reported catalytic enantioselective direct C-acylation of a ketone (Scheme II). The scope and limitations of this methodology were investigated and the products were successfully elaborated to afford a range of spirocyclic architectures. Finally, chapter 5 focuses on the kinetic analysis of the enantioselective C-acylation reaction in order to increase our understanding of the mechanisms behind phase-transfer catalysis and the mode of activation of the catalyst.</p