Duafalia F. Dudimah

Modulatory effects of bortezomib on host immune cell functions.

Bortezomib is an inhibitor of the ubiquitin-proteasome proteolytic pathway responsible for intracellular protein turnover. Cellular proteins controlled by this pathway represent a diverse group of potential therapeutic targets, particularly in cancer cells, which exploit this proteasomal pathway to promote their growth and diminish apoptosis. Along with inhibiting the proteasome and thus sensitizing tumor cells to apoptosis, bortezomib may also have multiple effects on the host immune responses. This review summarizes the effects that bortezomib may play on immune cell subsets in various disease states in modifying lymphocyte receptors, ligands, the expression of various cytokines and chemokines and their downstream signaling. We also propose steps that can be taken to refine combinatorial strategies that include bortezomib to improve current immunotherapeutic approaches.

Multivalent forms of the Notch ligand DLL-1 enhance antitumor T cell immunity in lung cancer and improve efficacy of EGFR targeted therapy

Activation of Notch signaling in hematopoietic cells by tumors contributes to immune escape. T-cell defects in tumors can be reversed by treating tumor-bearing mice with multivalent forms of the Notch receptor ligand DLL-1, but the immunologic correlates of this effect have not been elucidated. Here, we report mechanistic insights along with the efficacy of combinational treatments of multivalent DLL-1 with oncoprotein targeting drugs in preclinical mouse models of lung cancer. Systemic DLL-1 administration increased T-cell infiltration into tumors and elevated numbers of CD44(+)CD62L(+)CD8(+) memory T cells while decreasing the number of regulatory T cells and limiting tumor vascularization. This treatment was associated with upregulation of Notch and its ligands in tumor-infiltrating T cells enhanced expression of T-bet and phosphorylation of Stat1/2. Adoptive transfer of T cells from DLL1-treated tumor-bearing immunocompetent hosts into tumor-bearing SCID-NOD immunocompromised mice attenuated tumor growth and extended tumor-free survival in the recipients. When combined with the EGFR-targeted drug erlotinib, DLL-1 significantly improved progression-free survival by inducing robust tumor-specific T-cell immunity. In tissue culture, DLL1 induced proliferation of human peripheral T cells, but lacked proliferative or clonogenic effects on lung cancer cells. Our findings offer preclinical mechanistic support for the development of multivalent DLL1 to stimulate antitumor immunity.

Bortezomib enhances expression of effector molecules in anti-tumor CD8 + T lymphocytes by promoting Notch-nuclear factor-κB crosstalk

The immunosuppressive tumor microenvironment usurps host antitumor immunity by multiple mechanisms including interference with the Notch system, which is important for various metazoan cell fate decisions and hematopoietic cell differentiation and function. We observed that treatment with the proteasome inhibitor bortezomib in mice bearing various solid tumors resulted in an upregulated expression of various Notch signaling components in lymphoid tissues, thereby increasing CD8+T-lymphocyte IFNγ secretion and expression of effector molecules, perforin and granzyme B, as well as the T-box transcription factor eomesodermin. Bortezomib also neutralized TGFβ-mediated suppression of IFNγ and granzyme B expression in activated CD8+T-cells. Of note, bortezomib reversed tumor-induced downregulation of Notch receptors, Notch1 and Notch2, as well as increased the levels of cleaved Notch intracellular domain (NICD) and downstream targets Hes1 and Hey1 in tumor-draining CD8+T-cells. Moreover, bortezomib promoted CD8+T-cell nuclear factor-κB (NFκB) activity by increasing the total and phosphorylated levels of the IκB kinase and IκBα as well as the cytoplasmic and nuclear levels of phosphorylated p65. Even when we blocked NFκB activity by Bay-11-7082, or NICD cleavage by γ-secretase inhibitor, bortezomib significantly increased expression of Notch Hes1 and Hey1 genes as well as perforin, granzyme B and eomesodermin in activated CD8+T-cells. Data suggest that bortezomib can rescue tumor-induced dysfunction of CD8+T-cells by its intrinsic stimulatory effects promoting NICD-NFκB crosstalk. These findings provide novel insights on using bortezomib not only as an agent to sensitize tumors to cell death but also to provide lymphocyte-stimulatory effects, thereby overcoming immunosuppressive actions of tumor on anti-tumor T-cell functions.

Bortezomib improves adoptive T cell therapy by sensitizing cancer cells to FasL cytotoxicity

Cancer immunotherapy shows great promise but many patients fail to show objective responses, including in cancers that can respond well, such as melanoma and renal adenocarcinoma. The proteasome inhibitor bortezomib sensitizes solid tumors to apoptosis in response to TNF-family death ligands. Because T cells provide multiple death ligands at the tumor site, we investigated the effects of bortezomib on T-cell responses in immunotherapy models involving low-avidity antigens. Bortezomib did not affect lymphocyte or tissue-resident CD11c(+)CD8(+) dendritic cell counts in tumor-bearing mice, did not inhibit dendritic cell expression of costimulatory molecules, and did not decrease MHC class I/II-associated antigen presentation to cognate T cells. Rather, bortezomib activated NF-κB p65 in CD8(+) T cells, stabilizing expression of T-cell receptor CD3ζ and IL2 receptor-α, while maintaining IFNγ secretion to improve FasL-mediated tumor lysis. Notably, bortezomib increased tumor cell surface expression of Fas in mice as well as human melanoma tissue from a responsive patient. In renal tumor-bearing immunodeficient Rag2(-/-) mice, bortezomib treatment after adoptive T-cell immunotherapy reduced lung metastases and enhanced host survival. Our findings highlight the potential of proteasome inhibitors to enhance antitumor T-cell function in the context of cancer immunotherapy.

Bortezomib augments lymphocyte stimulatory cytokine signaling in the tumor microenvironment to sustain CD8 + T cell antitumor function

Tumor-induced immune tolerance poses a major challenge for therapeutic interventions aimed to manage cancer. We explored approaches to overcome T-cell suppression in murine breast and kidney adenocarcinomas, and lung fibrosarcoma expressing immunogenic antigens. We observed that treatment with a reversible proteasome inhibitor bortezomib (1 mg/kg body weight) in tumor-bearing mice significantly enhanced the expression of lymphocyte-stimulatory cytokines IL-2, IL-12, and IL-15. Notably, bortezomib administration reduced pulmonary nodules of mammary adenocarcinoma 4T1.2 expressing hemagglutinin (HA) model antigen (4T1HA) in mice. Neutralization of IL-12 and IL-15 cytokines with a regimen of blocking antibodies pre- and post-adoptive transfer of low-avidity HA518-526-specific CD8+T-cells following intravenous injection of 4T1HA cells increased the number of pulmonary tumor nodules. This neutralization effect was counteracted by the tumor metastasis-suppressing action of bortezomib treatments. In bortezomib-treated 4T1HA tumor-bearing mice, CD4+T-cells showed increased IL-2 production, CD11c+ dendritic cells showed increased IL-12 and IL-15 production, and HA-specific activated CD8+T-cells showed enhanced expression of IFNγ, granzyme-B and transcription factor eomesodermin. We also noted a trend of increased expression of IL-2, IL-12 and IL-15 receptors as well as increased phosphorylation of STAT5 in tumor-infiltrating CD8+T-cells following bortezomib treatment. Furthermore, bortezomib-treated CD8+T-cells showed increased phosphorylation of mitogen-activated protein kinase p38, and Akt, which was abrogated by phosphatidylinositide 3-kinase (PI3K) inhibitor. These data support the therapeutic potential of bortezomib in conjunction with other immunotherapies to augment the strength of convergent signals from CD8+T-cell signaling molecules including TCR, cytokine receptors and downstream PI3K/Akt/STAT5 pathways to sustain CD8+T-cell effector function in the tumor microenvironment.

Abstract 4161: Bortezomib enhances expression of effector molecules in antitumor CD8+ T lymphocytes by modulating Notch-NF-kB-miR-155 crosstalk

The immunosuppressive tumor microenvironment usurps host antitumor immunity by multiple mechanisms including interference with the Notch system, which is important for various metazoan cell fate decisions and hematopoietic cell differentiation and function. We observed that treatment with the proteasome inhibitor bortezomib in mice bearing various solid tumors resulted in an upregulated expression of various Notch signaling components in lymphoid tissues, thereby increasing CD8 + T-lymphocyte IFNγ secretion and expression of effector molecules, perforin and granzyme B, as well as the T-box transcription factor eomesodermin. Bortezomib also neutralized TGFβ-mediated suppression of IFNγ and granzyme B expression in activated CD8 + T-cells. Of note, bortezomib reversed tumor-induced downregulation of Notch receptors, Notch1 and Notch2, as well as increased the levels of cleaved Notch intracellular domain (NICD) and downstream targets Hes1 and Hey1 in tumor-draining CD8 + T-cells. Moreover, bortezomib promoted CD8 + T-cell nuclear factor-κB (NF-κB) activity by increasing the total and phosphorylated levels of the IκB kinase and IκBα as well as the cytoplasmic and nuclear levels of phosphorylated p65. Even when we blocked NFκB activity by Bay-11-7082, or NICD cleavage by γ-secretase inhibitor, bortezomib significantly increased expression of Notch Hes1 and Hey1 genes as well as perforin, granzyme B and eomesodermin in activated CD8 + T-cells. Data suggest that bortezomib can rescue tumor-induced dysfunction of CD8 + T-cells by its intrinsic stimulatory effects promoting NICD-NFκB crosstalk. We are also elucidating components of microRNA regulation affecting NICD-NFκB crosstalk. Our preliminary data suggest that miR-155 plays a role in bortezomib-induced regulation of T cell function. These findings provide novel insights on using bortezomib not only as an agent to sensitize tumors to cell death, but also to provide lymphocyte-stimulatory effects, thereby overcoming immunosuppressive actions of tumor on anti-tumor T-cell functions. Citation Format: Ariana N. Renrick, Menaka C. Thounaojam, Duafalia F. Dudimah, Portia Thomas, Samuel T. Pellom, Roman V. Uzhachenko, Anil Shanker. Bortezomib enhances expression of effector molecules in antitumor CD8+ T lymphocytes by modulating Notch-NF-kB-miR-155 crosstalk. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4161.

Bortezomib enhances anti-tumor T cell immunity by remodeling Notch system

The immunosuppressive tumor microenvironment perturbs numerous immune regulatory networks and usurps host antitumor immunity. We discovered that tumor interferes with host hematopoietic Notch system in lung cancer patients. The resultant decrease in immune Notch signaling could be a major causative link in the adequate induction of antitumor immunity. Interestingly, we observed that administration of the FDA- approved proteasome inhibitor drug Bortezomib (which also sensitizes tumors to death signals) to tumor bearing mice can restore Notch signaling in lymphoid cells without increasing tumor cell proliferation or clonogenicity. Moreover, Bortezomib administration altered Notch receptor and ligand expression pattern and increased the expression of Notch target genes Hes1, Hey1 and deltex1 in thymus, lymph node and spleen. Bortezomib administration in tumor bearing mice increased IFN-g production by T cells while the proportion of regulatory T cells was decreased. Our results indicate that the activation of Hes1 and Hey1 is mediated via inhibition of NFkB pathway while deltex1 activation is mediated by PI3K pathway. In another set of experiment, we observed that administration of Bortezomib along with adoptive CD8+ T cells transfer to tumor- bearing mice resulted in the reduction of tumor nodules, increased apoptosis and improve overall survival of mice. Our results clearly indicate that combining Bortezomib with adoptive T cell therapy can sustain T cell activation and function and, thus, enhances tumor immune surveillance. We are also elucidating a microRNA signature regulating immune Notch signaling. Our preliminary data suggest the role of miR-155 and miR-34a in Bortezomib induced regulation of T cell activation. The potential of Bortezomib to modulate anti-tumor Notch signaling and to enhance T cell activity presents exciting opportunities. Therapeutic restoration of immune Notch signaling by Bortezomib could help to break tumor resistance, enhance immune surveillance and sustain robust anti-tumor immunity.

Cancer therapy by resuscitating Notch immune surveillance

The immunosuppressive tumor microenvironment perturbs numerous immune regulatory networks and usurps host antitumor immunity. We discovered that tumor interferes with host hematopoietic Notch system in lung cancer patients [1]. The resultant decrease in immune Notch signaling could be a major causative link in the inadequate induction of antitumor immunity. Interestingly, administration of the novel Delta-like ligand 1 (DLL1) multivalent cluster [1] and the FDA-approved proteasome inhibitor drug bortezomib, which also sensitizes tumors to death signals [2,3], restored the tumor-induced decrease in immune Notch. Bortezomib increased the expression of Notch target genes Hes1 and Hey1 in thymus, lymph node, and spleen of tumor-bearing mice. Moreover, bortezomib administration decreased the proportion of regulatory T cells and enhanced antitumor T cell production of IFN-γ. Results indicate that bortezomib-induced activation of Notch target genes Hes1 and Hey1 is through its inhibition of NFκB while its activation of Deltex1 is mediated via PI3K. The potential of modulating antitumor Notch signaling by the prototypic DLL1 cluster in combination with bortezomib presents exciting opportunities to uncover multi-pronged immune stimulatory regimens. Therapeutic restoration of immune Notch signaling by bortezomib could provide effective treatment and recurrence-free survival in cancer patients by breaking tumor resistance, enhancing immune surveillance, and sustaining robust anti-tumor immunity.

Abstract 3642: Cancer therapy by resuscitating Notch immune surveillance

The immunosuppressive tumor microenvironment perturbs numerous immune regulatory networks and usurps host anti-tumor immunity. We discovered that tumor interferes with host hematopoietic Notch system in lung cancer patients. The resultant decrease in immune Notch signaling could be a major causative link in the inadequate induction of anti-tumor immunity. Interestingly, we found that tumor-induced decrease in immune Notch in various mouse solid tumor models could be restored therapeutically by the following two agents. Administration of a novel Delta-like ligand 1 (DLL1) multivalent cluster and the FDA-approved proteasome inhibitor drug bortezomib - which also sensitizes tumors to death signals - could activate Notch 1 signaling in lymphoid cells of tumor-bearing mice without increasing tumor cell proliferation or clonogenicity. Systemic activation of DLL1-Notch signaling could attenuate tumor vascularization as well as increase T cell infiltration in tumor, decrease proportion of regulatory T cells and enhance antitumor T cell function and memory in multiple mouse tumor models. New data also show that bortezomib affects the expression of notch receptors and ligands differentially in lymphocytes and in a wide range of solid tumor cells. Moreover, bortezomib administration increased the expression of Notch target genes Hes 1 and Hey 1 in thymus, lymph node, and spleen, as well as decreased the proportion of regulatory T cells and enhanced T cell production of IFN-γ in tumor-bearing mice. Results indicate that bortezomib-induced activation of Notch target genes Hes 1 and Hey 1 is through its inhibition of NFkB while its activation of another Notch target gene Deltex 1 is mediated via PI3K. The findings suggest a potential synergistic action of bortezomib and DLL1 activation of Notch signaling. The potential of modulating anti-tumor Notch signaling by the prototypic DLL1 cluster in combination with bortezomib presents exciting opportunities to uncover multi-pronged immune stimulatory regimens. Therapeutic restoration of immune Notch signaling could provide effective treatment and recurrence-free survival in cancer patients by breaking tumor resistance, enhancing immune surveillance, and sustaining robust anti-tumor immunity. Citation Format: Duafalia F. Dudimah, Samuel T. Pellom Jr., Roman V. Uzhachenko, David P. Carbone, Mikhail M. Dikov, Anil Shanker. Cancer therapy by resuscitating Notch immune surveillance. [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 3642. doi:10.1158/1538-7445.AM2014-3642

Cancer therapy by restoration of immune Notch

Factors affecting host immunity are important variables during cancer growth. The immunosuppressive tumor microenvironment perturbs numerous immune regulatory networks and usurps host antitumor immune responses. We discovered that tumor interferes with host hematopoietic Notch system. Tumor-induced decrease in Notch signaling in host immune cells could be a major causative link in the inadequate induction of anti-tumor immunity. Interestingly, we found that tumor-induced decrease in immune Notch could be restored therapeutically by the following two agents. Administration of a novel Delta-like ligand 1 (DLL1) multivalent cluster, and FDA-approved proteasome inhibitor drug bortezomib - which also sensitizes tumors to death signals - could activate Notch 1 signaling in lymphoid cells of tumor-bearing mice without increasing tumor cell proliferation or clonogenicity. Systemic activation of DLL1-Notch signaling could attenuate tumor vascularization as well as increase T cell infiltration in tumor, decrease proportion of regulatory T cells and enhance antitumor T cell function and memory in multiple mouse tumor models. This resulted in significant suppression of tumor growth in wild type mice as well as provided therapeutic benefit following an adoptive T cell transfer into tumor-bearing SCID-NOD mice. New data also show that bortezomib affects the expression of notch receptors and ligands differentially in lymphocytes and in a wide range of solid tumor cells: 4T1, PyMT, MDA-MB-231, C26, LLC, D459, and Renca-HA. Moreover, bortezomib administration increased the expression of Notch target Hes1 in thymus, lymph node and spleen of tumor-bearing mice, suggesting a potential synergistic action of bortezomib and DLL1 activation of Notch signaling. This new role of Notch in the development of effective antitumor immunity and the potential of its modulation by a novel prototypic agent DLL1 in combination with bortezomib present exciting opportunities to uncover multi-pronged immune stimulatory regimens. Reagents based on the multivalent forms of Notch ligands thus need to be explored for the therapeutic modulation of Notch signaling. Therapeutic restoration of immune Notch signaling could provide effective treatment and recurrence-free survival in cancer patients by breaking tumor resistance and induction of robust antitumor immunity.