CD155 blockade enhances allogeneic natural killer cell-mediated antitumor response against osteosarcoma.

Background: Osteosarcoma (OS) patients that present with metastatic disease have a poor prognosis and no curative options. Allogeneic bone marrow transplant (alloBMT) is curative for hematologic malignancies through the graft-versus-tumor (GVT) effect, but to date has been ineffective for solid tumors like OS. CD155 is expressed on OS and interacts strongly with the inhibitory receptors TIGIT and CD96 but also binds to the activating receptor DNAM-1 on natural killer (NK) cells but has never been targeted after alloBMT. Combining adoptive transfer of allogeneic NK (alloNK) cells with CD155 checkpoint blockade after alloBMT may enhance a GVT effect against OS but could enhance toxicities like graft-versus-host-disease (GVHD). Methods: Ex vivo activated and expanded murine NK cells were generated with soluble IL-15/IL- 15Rα. AlloNK and syngeneic NK (synNK) cell phenotype, cytotoxicity, cytokine production, and degranulation against the CD155-expressing murine OS cell line K7M2 were assessed in vitro. Mice bearing pulmonary OS metastases underwent alloBMT followed by infusion of alloNK cells with combinations of anti-CD155 and anti-DNAM-1 blockade. Tumor growth, GVHD and survival were monitored and differential gene expression of lung tissue was assessed by RNA microarray. Results: AlloNK cells exhibited superior cytotoxicity against CD155-expressing OS compared to synNK cells, and this activity was further enhanced by CD155 blockade. CD155 blockade increased alloNK cell degranulation and interferon gamma production through DNAM-1, as these functions were abrogated during DNAM-1 blockade. In vivo, CD155 blockade after alloBMT increased EFS with no exacerbation of GVHD. Treatment with combination CD155 and DNAM-1 blockade ameliorated survival and tumor control benefits seen with CD155 blockade alone. In mice treated with CD155 blockade, genes related to NK cell cytotoxicity were upregulated. DNAM-1 blockade resulted in upregulation of NK cell inhibition. Conclusions: These results demonstrate the safety and efficacy of infusing alloNK cells with CD155 blockade to mount a GVT effect against OS and show benefits are in part through DNAM-1. Defining the hierarchy of receptors that govern alloNK responses will be critical to translating combination adoptive NK cell and immune checkpoint inhibition for patients with solid tumors treated with alloBMT. WHAT IS ALREADY KNOWN ON THIS TOPIC: Allogeneic bone marrow transplant (alloBMT) has yet to show efficacy in treating solid tumors, such as osteosarcoma (OS). CD155 is expressed on OS and interacts with natural killer (NK) cell receptors, such as activating receptor DNAM-1 and inhibitory receptors TIGIT and CD96 and has a dominant inhibitory effect on NK cell activity. Targeting CD155 interactions on allogeneic NK cells could enhance anti-OS responses, but this has not been tested after alloBMT. WHAT THIS STUDY ADDS: CD155 blockade enhances allogeneic natural killer cell-mediated cytotoxicity against osteosarcoma and improved event-free survival after alloBMT in an in vivo mouse model of metastatic pulmonary OS. Addition of DNAM-1 blockade abrogated CD155 blockade-enhanced allogeneic NK cell antitumor responses. HOW THIS STUDY MIGHT AFFECT RESEARCH PRACTICE OR POLICY: These results demonstrate efficacy of allogeneic NK cells combined with CD155 blockade to mount an antitumor response against CD155-expressing OS. Translation of combination adoptive NK cell and CD155 axis modulation offers a platform for alloBMT treatment approaches for pediatric patients with relapsed and refractory solid tumors.

EGR1-mediated metabolic reprogramming to oxidative phosphorylation contributes to ibrutinib resistance in B-cell lymphoma.

The use of Bruton tyrosine kinase inhibitors, such as ibrutinib, to block B-cell receptor signaling has achieved a remarkable clinical response in several B-cell malignancies, including mantle cell lymphoma (MCL) and diffuse large B-cell lymphoma (DLBCL). Acquired drug resistance, however, is significant and affects the long-term survival of these patients. Here, we demonstrate that the transcription factor early growth response gene 1 (EGR1) is involved in ibrutinib resistance. We found that EGR1 expression is elevated in ibrutinib-resistant activated B-cell-like subtype DLBCL and MCL cells and can be further upregulated upon ibrutinib treatment. Genetic and pharmacological analyses revealed that overexpressed EGR1 mediates ibrutinib resistance. Mechanistically, TCF4 and EGR1 self-regulation induce EGR1 overexpression that mediates metabolic reprogramming to oxidative phosphorylation (OXPHOS) through the transcriptional activation of PDP1, a phosphatase that dephosphorylates and activates the E1 component of the large pyruvate dehydrogenase complex. Therefore, EGR1-mediated PDP1 activation increases intracellular adenosine triphosphate production, leading to sufficient energy to enhance the proliferation and survival of ibrutinib-resistant lymphoma cells. Finally, we demonstrate that targeting OXPHOS with metformin or IM156, a newly developed OXPHOS inhibitor, inhibits the growth of ibrutinib-resistant lymphoma cells both in vitro and in a patient-derived xenograft mouse model. These findings suggest that targeting EGR1-mediated metabolic reprogramming to OXPHOS with metformin or IM156 provides a potential therapeutic strategy to overcome ibrutinib resistance in relapsed/refractory DLBCL or MCL.

Plk1-Mediated Phosphorylation of TSC1 Enhances the Efficacy of Rapamycin.

The AKT/TSC/mTOR axis is an important pathway controlling cell growth, survival, and proliferation in response to extracellular cues. Recently, it was reported that AKT activity fluctuates across the cell cycle. However, it remains unclear whether downstream targets of AKT are also regulated by the cell cycle. Here, we report that mTORC1 activity inversely correlates with AKT activity during the cell cycle. Mechanistically, Plk1 phosphorylation of TSC1 at S467 and S578 interfered with TSC1/TSC2 binding, destabilized TSC1, promoted dissociation of the TSC complex from the lysosome, and eventually led to mTORC1 activation. Tumors derived from cancer cells expressing the TSC1-S467E/S578E mutant exhibited greater sensitivity to rapamycin than those expressing WT TSC1. Collectively, our data support a model in which Plk1, instead of AKT, regulates the TSC/mTORC1 pathway during mitosis, eventually regulating the efficacy of rapamycin.Significance: This seminal report shows that activation of mTORC1 can be independent of AKT during mitosis. Cancer Res; 78(11); 2864-75. ©2018 AACR.

CRR1 encoding callose synthase functions in ovary expansion by affecting vascular cell patterning in rice.

The ovary of rice undergoes rapid expansion immediately after fertilization, and this process determines the final sink strength potential of caryopses. To date, work on rice grain development has mainly focused on endosperm filling, whereas information on the essential elements for ovary expansion remains limited. We report here a functional analysis of the ovary expansion retarded mutant crr1 in rice. Map-based cloning revealed that CRR1 encodes a protein homologous to the Arabidopsis callose synthases AtGSL8 and AtGSL10. Point mutation in crr1 resulted in alternative splicing, which led to the formation of the truncated crr1 protein without the ß-glucan synthase domain. Iodine staining showed that there were few starch granules and these were unevenly distributed in the pericarp of crr1, and a 5,6-carboxyfluorescein diacetate transport assay revealed that carbohydrates were less efficiently unloaded from the lateral vasculature into the developing caryopsis. CRR1 transcripts were detected in all plant organs, with the highest level found in receptacles, which are mainly composed of vascular tissues. Analysis of pCRR1::GUS transgenic plants showed that CRR1 was specifically expressed in vascular bundle cells. Consistently, loss of function of CRR1 led to disordered patterns of vascular cells in the ovaries and receptacles of the mutant. Furthermore, a small portion of cells in the vascular bundles of crr1 showed defective cell wall formation, and callose deposition was specifically reduced at the plasmodesmata (PD) of cells with aberrant walls. Our results suggest that CRR1 performs a pivotal role in determining initial ovary expansion in rice, possibly via the PD-mediated permeability of cell fate determinants for vascular cell differentiation.