Mikhail M. Dikov

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.

Aquaporin 11 variant associates with kidney disease in type 2 diabetic patients.

Kidney disease, a common complication of diabetes, associates with poor prognosis. Our previous animal model studies linked aquaporin (AQP)11 to acute kidney injury, hyperglycemia-induced renal impairment, and kidney disease in diabetes. Here, we report the AQP11 rs2276415 variant as a genetic factor placing type 2 diabetic patients at greater risk for the development of kidney disease. We performed two independent retrospective case-control studies in 1,075 diabetic and 1,619 nondiabetic individuals who were identified in the Synthetic Derivative Database with DNA samples in the BioVU DNA repository at Vanderbilt University (Nashville, TN). A χ(2)-test and multivariable logistic regression analysis with adjustments for age, sex, baseline serum creatinine, and underlying comorbid disease covariates showed a significant association between rs2276415 and the prevalence of any event of acute kidney injury and chronic kidney disease (CKD) in diabetic patients but not in patients without diabetes. This result was replicated in the second independent study. Diabetic CKD patients over 55 yrs old with the minor AQP11 allele had a significantly faster progression of estimated glomerular filtration rate decline than patients with the wild-type genotype. Three-dimensional structural analysis suggested a functional impairment of AQP11 with rs2276415, which could place diabetic patients at a higher risk for kidney disease. These studies identified rs2276415 as a candidate genetic factor predisposing patients with type 2 diabetes to CKD.

Notch3-dependent β-catenin signaling mediates EGFR TKI drug persistence in EGFR mutant NSCLC

EGFR tyrosine kinase inhibitors cause dramatic responses in EGFR-mutant lung cancer, but resistance universally develops. The involvement of β-catenin in EGFR TKI resistance has been previously reported, however, the precise mechanism by which β-catenin activation contributes to EGFR TKI resistance is not clear. Here, we show that EGFR inhibition results in the activation of β-catenin signaling in a Notch3-dependent manner, which facilitates the survival of a subset of cells that we call “adaptive persisters”. We previously reported that EGFR-TKI treatment rapidly activates Notch3, and here we describe the physical association of Notch3 with β-catenin, leading to increased stability and activation of β-catenin. We demonstrate that the combination of EGFR-TKI and a β-catenin inhibitor inhibits the development of these adaptive persisters, decreases tumor burden, improves recurrence free survival, and overall survival in xenograft models. These results supports combined EGFR-TKI and β-catenin inhibition in patients with EGFR mutant lung cancer.Treatment of EGFR mutant non-small cell lung cancer (NSCLC) often develops resistance to EGFR TKIs. In this study, the authors discover a non-canonical activation of β-catenin signaling through Notch3 as a mechanism of adaptation to and resistance to EGFR TKI treatment in NSCLC.

Abstract 1624: NOTCH ligand-based therapeutics for immunomodulation in cancer and organ transplantation

We demonstrated in human and mouse studies that tumor-induced modulation of Notch ligand expression and Notch signaling in hematopoietic compartment contributes to tumor immune escape. Down-regulation of delta-like ligands (DLL) leads to defects in T cell development and T helper (Th1) cell differentiation with the prevalence of regulatory T cell (Treg) generation. To determine the roles of Notch ligands in antigen-presenting dendritic cells in regulation of antitumor immune responses we generated a set of lineage-specific knock-out mice lacking one of the Notch ligands in CD11c + dendritic cells (DC). We are developing and testing a set of reagents for clinical application for ligand-specific activation or inhibition of Notch signaling to stimulate or inhibit, respectively, various types of immune responses for applications in oncology and immune diseases. Mice with DLL1 insufficiency in DC demonstrated remarkably accelerated growth of Lewis lung carcinoma (LLC) tumor, and reduced survival compared to wild type animals. This associated with impaired anti-tumor immune responses indicated by the decreased tumor infiltration by IFNγ-producing T cells. Jagged2 knockout did not cause any significant alterations. Notch ligand expression in antigen-presenting cells was identified as a “checkpoint” regulating the type of immune response. Data reveal that expression of Notch ligands by antigen-presenting cells is an important immune response specifying mechanism and that ligand-specific Notch signaling could be a valuable therapeutic target. Reagents for the pharmacological modulation of immune responses based on Notch ligand constructs is proposed. Our cell-based study showed that pharmacological activation of Notch ligands required multivalent receptor-ligand interaction, whereas soluble ligands acted as competitive Notch inhibitors. We have generated reagents that comprise specific domains of the DLL1 in multivalent or monovalent form. Therapeutic inhibition of Notch by monovalent DLL1-based reagent accelerated LLC tumor growth and attenuated T cell-mediated anti-tumor immune response. In a heart transplantation mouse model, monovalent DLL1 reagent significantly prolonged allograft survival by inhibiting Th1 effector and memory T cell differentiation. Multivalent forms of DLL1 effectively stimulated Notch signaling in T cell cultures and enhanced IFNγ production, whereas monovalent reagent had opposite effects. Pharmacological up-regulation of DLL1-mediated Notch signaling with multivalent forms of ligand represents an efficient strategy for the enhancement of anti-tumor immunity and targeting multiple mechanisms of tumor growth. Monovalent DLL1 forms could be utilized for therapeutic inhibition of Th1 responses in autoimmune diseases and organ transplantation. Reagents based on the mono- and multivalent forms of Notch ligands can be efficiently utilized for therapeutic modulation of Notch signaling. Note: This abstract was not presented at the meeting. Citation Format: Elena I. Tchekneva, Anneliese E. Antonucci, Irina Chekneva, Nicholas Long, Jason V. Evans, Anwari Akhter, David P. Carbone, Thomas Magliery, Mikhail M. Dikov. NOTCH ligand-based therapeutics for immunomodulation in cancer and organ transplantation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1624. doi:10.1158/1538-7445.AM2017-1624

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

Abstract 1177: Pharmacological stimulation of DLL1-Notch signaling as an effective cancer immunotherapy

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Our data demonstrated that in cancer, expression of Notch ligands is selectively altered and Notch signaling in the hematopoietic compartment is attenuated and associated with impaired anti-tumor immunity caused by the aberrant T cell differentiation and function. Our important and therapeutically relevant finding is that stimulation of DLL1-mediated Notch signaling by systemic administration of soluble multivalent (clustered) DLL1 efficiently restores Notch signaling in the hematopoietic compartment. Such therapy strongly activates Notch downstream signaling and variably modulates expression of Notch receptors and ligands. Clustered DLL1 treatment results in significant enhancement of tumor antigen-specific T cell immune responses and memory, decreased proportion of regulatory T cells, remarkably increased tumor infiltration by T cells, attenuated tumor vascularization, and suppression of tumor growth. In an inducible model of lung tumor expressing L858R mutant EGFR in lung epithelium, clustered DLL1 elicited robust anti-tumor immune responses in concert with EGFR inhibition by erlotinib and produced remarkably improved progression-free survival. It is also likely that more efficient tumor killing and induction of T cell memory would significantly delay tumor progression/recurrence. Activation of Notch signaling by multivalent DLL1 in a panel of lung cancer cells in vitro did not stimulate cells proliferative of colony-forming potential, but rather demonstrated inhibitory effect in several cases. Pharmacological stimulation of Notch by multivalent DLL1 might represent an efficient strategy for enhancement of anti-tumor immunity and targeting multiple mechanisms of tumor growth. Reagents based on the multivalent forms of Notch ligands can be efficiently utilized for the therapeutic modulation of Notch signaling. Citation Format: Asel K. Biktasova, Fred D. Dudimah, Roman Uzhachenko, Rajeswara Rao Arasada, Elena E. Tchekneva, David P. Carbone, Anil Shanker, Mikhail M. Dikov. Pharmacological stimulation of DLL1-Notch signaling as an effective cancer immunotherapy. [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 1177. doi:10.1158/1538-7445.AM2014-1177