Immunomodulatory drugs downregulate IKZF1 leading to expansion of hematopoietic progenitors with concomitant block of megakaryocytic maturation.

The immunomodulatory drugs, lenalidomide and pomalidomide yield high response rates in multiple myeloma patients, but are associated with a high rate of thrombocytopenia and increased risk of secondary hematologic malignancies. Here, we demonstrate that the immunomodulatory drugs induce self-renewal of hematopoietic progenitors and upregulate megakaryocytic colonies by inhibiting apoptosis and increasing proliferation of early megakaryocytic progenitors via down-regulation of IKZF1. In this process, the immunomodulatory drugs degrade IKZF1 and subsequently down-regulate its binding partner, GATA1. This results in the decrease of GATA1 targets such as ZFPM1 and NFE2, leading to expansion of megakaryocytic progenitors with concomitant inhibition of maturation of megakaryocytes. The down-regulation of GATA1 further decreases CCND1 and increases CDKN2A expression. Overexpression of GATA1 abrogated the effects of the immunomodulatory drugs and restored maturation of megakaryocytic progenitors. Our data not only provide the mechanism for the immunomodulatory drugs induced thrombocytopenia but also help to explain the higher risk of secondary malignancies and long-term cytopenia induced by enhanced cell cycling and subsequent exhaustion of the stem cell pool.

Interaction between Her2 and Beclin-1 proteins underlies a new mechanism of reciprocal regulation.

Beclin-1 is a key regulator of autophagy that functions in the context of two phase-specific complexes in the initiation and maturation of autophagosomes. Its known interacting proteins include autophagy effectors, Bcl-2 family members, and organelle membrane anchor proteins. Here we report a newly identified interaction between Beclin-1 and the protein tyrosine kinase receptor Her2. We demonstrate that in Her2-expressing breast carcinoma cells that do not succumb to lapatinib, this Her1/2 inhibitor disrupts the cell surface interaction between Her2 and Beclin-1. The data suggest that the ensuing autophagic response is correlatively associated with the release of Beclin-1 from its complex with Her2 and with the subsequent increase in cytosolic Beclin-1. Upon its interaction with Her2, Beclin-1 up-regulates the phosphorylation levels of Her2 and Akt. The Beclin-1 evolutionarily conserved domain is required both for the interaction of Beclin-1 with Her2 and for the increased Her2 and Akt phosphorylation. These findings shed new light on mechanisms involved in lapatinib-mediated autophagy in Her2-expressing breast carcinoma cell lines and in Beclin-1 signaling in these cells.

IFN-γR/STAT1 signaling in recipient hematopoietic antigen-presenting cells suppresses graft-versus-host disease.

The absence of IFN-γ receptor (IFN-γR) or STAT1 signaling in donor cells has been shown to result in reduced induction of acute graft-versus-host disease (GVHD). In this study, we unexpectedly observed increased activation and expansion of donor lymphocytes in both lymphohematopoietic organs and GVHD target tissues of IFN-γR/STAT1-deficient recipient mice, leading to rapid mortality following the induction of GVHD. LPS-matured, BM-derived Ifngr1-/- Stat1-/- DCs (BMDCs) were more potent allogeneic stimulators and expressed increased levels of MHC II and costimulatory molecules. Similar effects were observed in human antigen-presenting cells (APCs) with knockdown of Stat1 by CRISPR/Cas9 and treatment with a JAK1/2 inhibitor. Furthermore, we demonstrated that the absence of IFN-γR/STAT1 signaling in hematopoietic APCs impaired the presentation of exogenous antigens, while promoting the presentation of endogenous antigens. Thus, the indirect presentation of host antigens to donor lymphocytes was defective in IFN-γR/STAT1-deficient, donor-derived APCs in fully donor chimeric mice. The differential effects of IFN-γR/STAT1 signaling on endogenous and exogenous antigen presentation could provide further insight into the roles of the IFN-γ/STAT1 signaling pathway in the pathogenesis of GVHD, organ rejection, and autoimmune diseases.

Tumor-resident regulatory T cells in pancreatic cancer express the αvß5 integrin as a targetable activation marker.

Pancreatic ductal adenocarcinoma (PDAC) has abundant immunosuppressive regulatory T cells (Tregs), which contribute to a microenvironment resistant to immunotherapy. Here, we report that Tregs in the PDAC tissue, but not those in the spleen, express the αvß5 integrin in addition to neuropilin-1 (NRP-1), which makes them susceptible to the iRGD tumor-penetrating peptide, which targets cells positive for αv integrin- and NRP-1. As a result, long-term treatment of PDAC mice with iRGD leads to tumor-specific depletion of Tregs and improved efficacy of immune checkpoint blockade. αvß5 integrin + Tregs are induced from both naïve CD4 + T cells and natural Tregs upon T cell receptor stimulation, and represent a highly immunosuppressive subpopulation of CCR8 + Tregs. This study identifies the αvß5 integrin as a marker for activated tumor-resident Tregs, which can be targeted to achieve tumor-specific Treg depletion and thereby augment anti-tumor immunity for PDAC therapy.

Low prevalence of hepatitis B virus infection in patients with systemic lupus erythematosus in southern China.

To investigate the prevalence of hepatitis B virus (HBV) infection in systemic lupus erythematosus (SLE) patients in southern China, SLE inpatients were retrospectively investigated for their HBV infection rate. Fifteen SLE patients positive for hepatitis B surface antigen (HBsAg) were followed up. Furthermore, serum interferon (IFN)-α levels among SLE patients were detected by ELISA. Results showed estimated HBsAg-positive rate was 10.74% in general population. The HBsAg-positive rate was lower in SLE patients compared with controls (2.33 vs. 9.57%, P < 0.01). Interestingly, 13 out of 15 SLE patients converted from HBsAg positive to HBV surface antibody (HBsAb) positive even under glucocorticoid therapy. In addition, we found significantly increased IFN-α levels in SLE patients.The prevalence of HBV infection in SLE patients was lower than that in sex- and age-matched non-SLE controls in southern China. The characteristic IFN signatures in SLE may favor the subsequent clearance of HBV.

Sinomenine suppresses TNF-alpha-induced VCAM-1 expression in human umbilical vein endothelial cells.

Sinomenine (SN), an alkaloid prepared from the root of Sinomenium acutum Rehd. Et wils, is used to alleviate the symptoms of rheumatism in Chinese medicine. In the present study, the potential inhibition of TNF-alpha-induced VCAM-1 expression on human umbilical vein endothelial cells (HUVECs) was evaluated in vitro. HUVECs were isolated from freshly collected umbilical cords. Positive controls were stimulated with TNF-alpha, omitting SN. Negative controls were cultured omitting TNF-alpha and SN. Experimental groups were co-cultured with TNF-alpha and SN at different concentrations (0.25, 0.5, and 1.0 mol/L), or TNF-alpha and Dexamethasone (Dex) at a concentration of 1.0 x 10(-6) mol/L. Cells were harvested after culturing with the above drugs for 12 h. VCAM-1 mRNA expression was detected by real-time quantitative PCR, and VCAM-1 expression was detected by flow cytometry. The experimental data indicated that VCAM-1 mRNA and VCAM-1 were induced by TNF-alpha. The relative VCAM-1 mRNA expression decreased in the experimental groups (p<0.05). Concentrations of SN at 0.5 and 1.0 mol/L inhibited expression of VCAM-1 (p<0.05). SN at concentration of 0.25 mol/L and Dex at concentration of 1.0 x 10(-6) mol/L did not show an inhibitory effect on VCAM-1 expression in TNF-alpha-induced HUVECs. Our preliminary data indicates that SN has an inhibitory effect in vitro on TNF-alpha-induced VCAM-1 expression at both mRNA level and protein level in HUVECs, and suggests that SN may be a novel method of immunotherapy for rheumatic carditis or rheumatic heart disease.

Elevated Translation Initiation Factor eIF4E Is an Attractive Therapeutic Target in Multiple Myeloma.

eIF4E is the key regulator of protein translation and critical for translation. The oncogenic potential of tumorigenesis, which is highly contingent on cap-dependent eIF4E, also arises from the critical role in the nuclear export and cytosolic translation of oncogenic transcripts. Inhibition of Exportin1 (XPO1), which is the major nuclear export protein for eIF4E-bound oncoprotein mRNAs, results in decreased tumor cell growth in vitro and in vivo, suggesting that eIF4E is critical in multiple myeloma. Indeed, we found that eIF4E is overexpressed in myeloma cell lines and primary myeloma cells compared with normal plasma cells. Although stable overexpression of eIF4E in multiple myeloma cells significantly increases tumorigenesis, knockdown of eIF4E impairs multiple myeloma tumor progression in a human xenograft mouse model. Using a tet-on-inducible eIF4E-knockdown system, eIF4E downregulation blocks multiple myeloma tumor growth in vivo, correlating with decreased eIF4E expression. Further overexpression and knockdown of eIF4E revealed that eIF4E regulates translation of mRNAs with highly complex 5'-untranslated regions, such as c-MYC and C/EBPß, and subsequently proliferation in multiple myeloma cells, but not in nonmalignant bone marrow stromal cells. Because many transcription factors that are critical for multiple myeloma proliferation exhibit a higher dependency on protein translation, eIF4E is an ideal and selective tool to target multiple myeloma cell growth. Mol Cancer Ther; 15(4); 711-9. ©2016 AACR.

Absence of Stat1 in donor CD4⁺ T cells promotes the expansion of Tregs and reduces graft-versus-host disease in mice.

STAT1 is the main signal transducer for type I and II IFNs and plays a central role in the regulation of innate and adaptive immune responses. We used Stat1-deficient mice to test the role of donor Stat1 in MHC-matched minor histocompatibility antigen-mismatched (mHA-mismatched) and fully MHC-mismatched models of bone marrow transplantation. Lack of Stat1 in donor splenocytes reduced graft-versus-host disease (GVHD) in both immunogenetic disparities, leading to substantially attenuated morbidity and mortality. Donor Stat1 deficiency resulted in reduced alloantigen-induced activation and expansion of donor T cells and correlated with the expansion of CD4+CD25+Foxp3+ Tregs in vivo. This expansion of Tregs was further confirmed by studies showing that Stat1 deficiency promoted the proliferation, while inhibiting the apoptosis, of natural Tregs, and that absence of Stat1 enhanced the induction of inducible Tregs both in vitro and in vivo. Ex vivo expanded Stat1-/- Tregs were superior to wild-type Tregs in suppressing alloantigen-driven expansion of T cells in vitro and in inhibiting the development of GVHD. These observations demonstrate that Stat1 is a regulator of Tregs and that targeting Stat1 in CD4+ T cells may facilitate in vitro and in vivo expansion of Tregs for therapeutic use.

Targeting the microtubular network as a new antimyeloma strategy.

We identified nocodazole as a potent antimyeloma drug from a drug screening library provided by the Multiple Myeloma Research Foundation. Nocodazole is a benzimidazole that was originally categorized as a broad-spectrum anthelmintic drug with antineoplastic properties. We found that nocodazole inhibited growth and induced apoptosis of primary and multiresistant multiple myeloma cells cultured alone and in the presence of bone marrow stromal cells. Nocodazole caused cell-cycle prophase and prometaphase arrest accompanied by microtubular network disarray. Signaling studies indicated that increased expression of Bim protein and reduced X-linked inhibitor of apoptosis protein and Mcl-1(L) levels were involved in nocodazole-induced apoptosis. Further investigation showed Bcl-2 phosphorylation as a critical mediator of cell death, triggered by the activation of c-jun-NH(2) kinase (JNK) instead of p38 kinase or extracellular signal-regulated kinases. Treatment with JNK inhibitor decreased Bcl-2 phosphorylation and subsequently reduced nocodazole-induced cell death. Nocodazole combined with dexamethasone significantly inhibited myeloma tumor growth and prolonged survival in a human xenograft mouse model. Our studies show that nocodazole has potent antimyeloma activity and that targeting the microtubular network might be a promising new treatment approach for multiple myeloma.

Autophagic degradation of active caspase-8: a crosstalk mechanism between autophagy and apoptosis.

Apoptotic defects endow tumor cells with survival advantages. Such defects allow the cellular stress response to take the path of cytoprotective autophagy, which either precedes or effectively blocks an apoptotic cascade. Inhibition of the cytoprotective autophagic response shifts the cells toward apoptosis, by interfering with an underlying molecular mechanism of cytoprotection. The current study has identified such a mechanism that is centered on the regulation of caspase-8 activity. The study took advantage of Bax(-/-) Hct116 cells that are TRAIL-resistant despite significant DISC processing of caspase-8, and of the availability of a caspase-8-specific antibody that exclusively detects the caspase-8 large subunit or its processed precursor. Utilizing these biological tools, we investigated the expression pattern and subcellular localization of active caspase-8 in TRAIL-mediated autophagy and in the autophagy-to-apoptosis shift upon autophagy inhibition. Our results suggest that the TRAIL-mediated autophagic response counter-balances the TRAIL-mediated apoptotic response by the continuous sequestration of the large caspase-8 subunit in autophagosomes and its subsequent elimination in lysosomes. The current findings are the first to provide evidence for regulation of caspase activity by autophagy and thus broaden the molecular basis for the observed polarization between autophagy and apoptosis.

Enhancement of tumor-TRAIL susceptibility by modulation of autophagy.

Autophagy has recently been recognized as an important cellular response to stress. However, the prospect of manipulating the autophagic process for the enhancement of cancer therapy remains unresolved. This lack of resolution stems from the current controversy regarding the fundamental function of autophagy in tumor stress response: Does it have a positive or negative impact on tumor survival capability? Our studies were designed to investigate the role of autophagy in the response to TRAIL of tumor cells with various apoptotic defects. Based on our findings, we propose that divergent mechanisms of resistance to TRAIL can be reversed by a common approach of targeting specific components of the autophagic process for inhibition. This concept may have significant implications for the development of new strategies to circumvent TRAIL resistance in tumors.

Involvement of protective autophagy in TRAIL resistance of apoptosis-defective tumor cells.

Targeting TRAIL receptors with either recombinant TRAIL or agonistic DR4- or DR5-specific antibodies has been considered a promising treatment for cancer, particularly due to the preferential apoptotic susceptibility of tumor cells over normal cells to TRAIL. However, the realization that many tumors are unresponsive to TRAIL treatment has stimulated interest in identifying apoptotic agents that when used in combination with TRAIL can sensitize tumor cells to TRAIL-mediated apoptosis. Our studies suggest that various apoptosis defects that block TRAIL-mediated cell death at different points along the apoptotic signaling pathway shift the signaling cascade from default apoptosis toward cytoprotective autophagy. We also obtained evidence that inhibition of such a TRAIL-mediated autophagic response by specific knockdown of autophagic genes initiates an effective mitochondrial apoptotic response that is caspase-8-dependent. Currently, the molecular mechanisms linking disabled autophagy to mitochondrial apoptosis are not known. Our analysis of the molecular mechanisms involved in the shift from protective autophagy to apoptosis in response to TRAIL sheds new light on the negative regulation of apoptosis by the autophagic process and by some of its individual components.

Identification of polyclonal and monoclonal antibodies against tissue plasminogen activator in the antiphospholipid syndrome.

OBJECTIVE: To test the hypotheses that some plasmin-reactive anticardiolipin antibodies (aCL) may bind to tissue plasminogen activator (tPA) and that some of the tPA-reactive aCL may inhibit tPA activity. METHODS: We studied the reactivity of 8 patient-derived monoclonal aCL with tPA and examined the presence of IgG anti-tPA antibodies in patients with the antiphospholipid syndrome (APS). The effects of the reactive monoclonal aCL on the activity of tPA were also examined. RESULTS: Six patient-derived plasmin-reactive monoclonal aCL bound to tPA. Analysis of plasma samples revealed that 10 of 80 APS patients (12.5%) and 1 of 81 systemic lupus erythematosus patients (1.2%) had antibodies against fibrin-associated tPA, based on a cutoff value equal to the mean + 2SD of the level in 28 normal subjects. Of the 6 tPA-reactive monoclonal aCL, 2 of them (CL1 and CL15) inhibited tPA activity. CONCLUSION: Some of the plasmin-reactive aCL in APS patients may bind to tPA. Of the tPA-reactive aCL, some (such as CL1 and CL15) may inhibit tPA activity and, thus, may be prothrombotic in the host.