Asel Biktasova

Adenosinergic Regulation of the Expansion and Immunosuppressive Activity of CD11b+Gr1+ Cells

Extracellular adenosine and purine nucleotides are elevated in many pathological situations associated with the expansion of CD11b+Gr1+ myeloid-derived suppressor cells (MDSCs). Therefore, we tested whether adenosinergic pathways play a role in MDSC expansion and functions. We found that A2B adenosine receptors on hematopoietic cells play an important role in accumulation of intratumoral CD11b+Gr1high cells in a mouse Lewis lung carcinoma model in vivo and demonstrated that these receptors promote preferential expansion of the granulocytic CD11b+Gr1high subset of MDSCs in vitro. Flow cytometry analysis of MDSCs generated from mouse hematopoietic progenitor cells revealed that the CD11b+Gr-1high subset had the highest levels of CD73 (ecto-5′-nucleotidase) expression (Δmean fluorescence intensity [MFI] of 118.5 ± 16.8), followed by CD11b+Gr-1int (ΔMFI of 57.9 ± 6.8) and CD11b+Gr-1−/low (ΔMFI of 12.4 ± 1.0) subsets. Even lower levels of CD73 expression were found on Lewis lung carcinoma tumor cells (ΔMFI of 3.2 ± 0.2). The high levels of CD73 expression in granulocytic CD11b+Gr-1high cells correlated with high levels of ecto-5′-nucleotidase enzymatic activity. We further demonstrated that the ability of granulocytic MDSCs to suppress CD3/CD28-induced T cell proliferation was significantly facilitated in the presence of the ecto-5′-nucleotidase substrate 5′-AMP. We propose that generation of adenosine by CD73 expressed at high levels on granulocytic MDSCs may promote their expansion and facilitate their immunosuppressive activity.

Aquaporin 11 insufficiency modulates kidney susceptibility to oxidative stress.

Aquaporin 11 (AQP11) is a newly described member of the protein family of transport channels. AQP11 associates with the endoplasmic reticulum (ER) and is highly expressed in proximal tubular epithelial cells in the kidney. Previously, we identified and characterized a recessive mutation of the highly conserved Cys227 to Ser227 in mouse AQP11 that caused proximal tubule (PT) injury and kidney failure in mutant mice. The current study revealed induction of ER stress, unfolded protein response, and apoptosis as molecular mechanisms of this PT injury. Cys227Ser mutation interfered with maintenance of AQP11 oligomeric structure. AQP11 is abundantly expressed in the S1 PT segment, a site of major renal glucose flux, and Aqp11 mutant mice developed PT-specific mitochondrial injury. Glucose increased AQP11 protein expression in wild-type kidney and upregulation of AQP11 expression by glucose in vitro was prevented by phlorizin, an inhibitor of sodium-dependent glucose transport across PT. Total AQP11 levels in heterozygotes were higher than in wild-type mice but were not further increased in response to glucose. In Aqp11 insufficient PT cells, glucose potentiated increases in reactive oxygen species (ROS) production. ROS production was also elevated in Aqp11 mutation carriers. Phenotypically normal mice heterozygous for the Aqp11 mutation repeatedly treated with glucose showed increased blood urea nitrogen levels that were prevented by the antioxidant sulforaphane or by phlorizin. Our results indicate an important role for AQP11 to prevent glucose-induced oxidative stress in proximal tubules.

Role of JunB in adenosine A2B receptor-mediated vascular endothelial growth factor production.

Interstitial adenosine stimulates neovascularization in part through A2B adenosine receptor-dependent upregulation of vascular endothelial growth factor (VEGF). In the current study, we tested the hypothesis that A2B receptors upregulate JunB, which can contribute to stimulation of VEGF production. Using the human microvascular endothelial cell line, human mast cell line, mouse cardiac Sca1-positive stromal cells, and mouse Lewis lung carcinoma (LLC) cells, we found that adenosine receptor-dependent upregulation of VEGF production was associated with an increase in VEGF transcription, activator protein-1 (AP-1) activity, and JunB accumulation in all cells investigated. Furthermore, the expression of JunB, but not the expression of other genes encoding transcription factors from the Jun family, was specifically upregulated. In LLC cells expressing A2A and A2B receptor transcripts, only the nonselective adenosine agonist NECA (5′-N-ethylcarboxamidoadenosine), but not the selective A2A receptor agonist CGS21680 [2-p-(2-carboxyethyl) phenylethylamino-5′-N-ethylcarboxamidoadenosine], significantly increased JunB reporter activity and JunB nuclear accumulation, which were inhibited by the A2B receptor antagonist PSB603 [(8-[4-[4-((4-chlorophenzyl)piperazide-1-sulfonyl)phenyl]]-1-propylxanthine]. Using activators and inhibitors of intracellular signaling, we demonstrated that A2B receptor-dependent accumulation of JunB protein and VEGF secretion share common intracellular pathways. NECA enhanced JunB binding to the murine VEGF promoter, whereas mutation of the high-affinity AP-1 site (−1093 to −1086) resulted in a loss of NECA-dependent VEGF reporter activity. Finally, NECA-dependent VEGF secretion and reporter activity were inhibited by the expression of a dominant negative JunB or by JunB knockdown. Thus, our data suggest an important role of the A2B receptor-dependent upregulation of JunB in VEGF production and possibly other AP-1–regulated events.

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.

Demethylation therapy as a targeted treatment for human papilloma virus-associated head and neck cancer

Purpose: DNA methylation in human papillomavirus–associated (HPV+) head and neck squamous cell carcinoma (HNSCC) may have importance for continuous expression of HPV oncogenes, tumor cell proliferation, and survival. Here, we determined activity of a global DNA-demethylating agent, 5-azacytidine (5-aza), against HPV+ HNSCC in preclinical models and explored it as a targeted therapy in a window trial enrolling patients with HPV+ HNSCC. Experimental Design: Sensitivity of HNSCC cells to 5-aza treatment was determined, and then 5-aza activity was tested in vivo using xenografted tumors in a mouse model. Finally, tumor samples from patients enrolled in a window clinical trial were analyzed to identify activity of 5-aza therapy in patients with HPV+ HNSCC. Results: Clinical trial and experimental data show that 5-aza induced growth inhibition and cell death in HPV+ HNSCC. 5-aza reduced expression of HPV genes, stabilized p53, and induced p53-dependent apoptosis in HNSCC cells and tumors. 5-aza repressed expression and activity of matrix metalloproteinases (MMP) in HPV+ HNSCC, activated IFN response in some HPV+ head and neck cancer cells, and inhibited the ability of HPV+ xenografted tumors to invade mouse blood vessels. Conclusions: 5-aza may provide effective therapy for HPV-associated HNSCC as an alternative or complement to standard cytotoxic therapy. Clin Cancer Res; 23(23); 7276–87. ©2017 AACR.

Abstract 1175: Inhibition of NAMPT as a novel therapeutic strategy for infant leukemia

Novel targeted therapies are urgently needed for infant leukemia as this disease is highly aggressive and refractory to treatment resulting in poor survival rates. The NAD (Nicotinamide Adenine Dinucleotide) producing enzyme NAMPT (Nicotinamide Phosphoribosyltransferase) has been considered an attractive selective target for anti-cancer therapy due to the high dependency of tumor cells on NAD for energy metabolism and activity of NAD-dependent enzymes such as poly-ADP-ribose polymerases (PARPs) and sirtuins that play key roles in cancer cell survival. A newly developed NAMPT inhibitor, OT-82, was initially isolated for its high selective toxicity against a panel of adult leukemia cell lines. Here we investigated NAMPT inhibition as a therapeutic strategy for infant leukemias characterized by rearrangement of the MLL gene (MLL-r), by testing the potency of OT-82 in a panel of preclinical in vitro and in vivo models of MLL-r leukemia that are based on the use of patient-derived xenograft (PDX) cells. OT-82, as a single agent, dramatically reduced the viability of all tested MLL-r leukemia cell lines (n=9) and MLL-r leukemia PDX (n=6) with IC50s ranging from 0.15 to 3.82nM. While the IC50 for OT-82 correlated significantly with the IC50 of other NAMPT inhibitors STF-118804 and FK866, OT-82 was the most potent compound. When combining OT-82 with chemotherapeutic agents currently used to treat infants with leukemia, we observed significant synergy between OT-82 and cytarabine indicating the potential of OT-82 for chemosensitization. Consistent with NAMPT inhibition, OT-82 reduced cytosolic NAD+ levels in MLL-r leukemia cells and inhibited the activity of the NAD-requiring enzymes PARP-1 and SIRT-1, as exemplified by a decrease in PARylated PARP-1 levels and a p53-mediated increase in p21 levels, leading to apoptosis induction. Interestingly, despite the remarkable potency of OT-82 in killing MLL-r leukemia cells, a 25-fold difference in IC50 levels was noted across the cell line panel, with those lines harboring the MLL translocations most prevalent in infants, namely t(4;11) and t(11;19) translocations, being the most sensitive to the compound. A positive correlation was noted between baseline NAMPT mRNA levels and OT-82 IC50 (r=0.7712, P=0.015). Further in vivo testing of OT-82 showed impressive efficacy of the compound in MLL-r leukemia PDX-based animal models (n=6). OT-82 (p.o. 3x week, 40-50 mg/kg, for 3 or 6 weeks) was well tolerated and significantly delayed leukemia progression in 6/6 MLL-r leukemia xenografts with 5/6 achieving objective responses. OT-82 given as monotherapy was as effective as the routinely used triple combination treatment of vincristine, L-asparaginase and dexamethasone. Overall, these results demonstrate that NAMPT inhibition using OT-82 is highly effective against MLL-r leukemia and when combined with current chemotherapies may offer a more selective and potent therapeutic option for infants suffering from this disease. Citation Format: Klaartje Somers, Shiloh Middlemiss, Asel Biktasova, Mawar Karsa, Leanna Cheung, Angelika Kosciolek, Kathryn Evans, Chelsea Mayoh, Ursula R. Kees, Lioubov Korotchkina, Olga B. Chernova, Richard B. Lock, Andrei V. Gudkov, Michelle Haber, Murray D. Norris, Michelle J. Henderson. Inhibition of NAMPT as a novel therapeutic strategy for infant leukemia [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 1175. doi:10.1158/1538-7445.AM2017-1175

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.

Abstract 3983: Resuscitating immune surveillance in cancer.

Cancer growth interferes with the Notch signaling that plays diverse roles in lymphocyte commitment and differentiation, oncogenesis, and peripheral immune responses. Our studies show that the stimulation of hematopoietic Notch signaling following administration of a prototypic therapeutic agent clustered multivalent Delta-like Notch ligand (DLL1), in mouse lung tumor models can correct tumor-induced defects in lymphocyte differentiation and improve antitumor lymphocyte responses, without increasing tumor cell proliferation. Moreover, since the Notch signaling complex is inactivated via the proteasome, its inhibitor bortezomib, amongst its other potential effects, may sustain DLL1-mediated pharmacological activation of the Notch signaling. Our data demonstrate that the proteasome inhibitor bortezomib can selectively sensitize solid tumors to apoptotic signals by upregulating death receptors on tumor cells and amplifying caspase-8 activation in the extrinsic cell death pathway, without any adverse effect on the antigen-specific T cell proliferative or cytolytic functions in an in vivo setting. In the current study, we investigated the effects of bortezomib and DLL1 on Notch receptor/ligand system and its downstream signaling network with an aim to enhance cancer immunotherapy. Results 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. Specifically, the expression of Notch 4 receptor and its ligand Jagged 1 was reduced in bortezomib-treated tumor cells. In addition, bortezomib treatment of tumor cells modulated Notch downstream targets Hes1, Hes3, Hey1, Deltex1 and Deltex3, which act as transcriptional regulators of tissue-specific differentiation. bortezomib also modulated the phosphorylation of Erk1/2 MAP kinases and AKT suggesting that bortezomib may mediate its effects through downstream phosphorylation of Erk targets, such as STATs and NFkB, or PI3K, which are key players in signaling cross-talk and intersect with Notch signaling. Noreover, 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. These results suggest that tumor cell-death sensitizing bortezomib and pharmacological DLL1 cluster activating immunostimulatory Notch in tumor-bearing host follow common signal transduction pathways and can synergize in antitumor activity. These findings offer a novel three-pronged combinatorial therapeutic approach of immunostimulatory Notch ligand DLL1 and bortezomib to abrogate the immunosuppressive circuitries operating in the tumor microenvironment with a potential to reduce tumor burden and resuscitate tumor-specific immunity. Citation Format: Duafalia F. Dudimah, Roman V. Uzhachenko, Samuel T. Pellom, Asel K. Biktasova, Mikhail M. Dikov, David P. Carbone, Anil Shanker. Resuscitating immune surveillance in cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3983. doi:10.1158/1538-7445.AM2013-3983

Abstract A9: Pharmacological stimulation of DLL1-Notch signaling as an antitumor immunotherapy

Deficiencies in immune function are one of mechanisms for tumor escape from host immune surveillance, and therapies aimed at overcoming these defects are of major importance in cancer immunotherapy. However, the underlying causes of these defects are multiple and remain not fully understood. We report a previously undescribed mechanism for tumor-associated defects in T cell function mediated by reducing levels of Delta-like (DLL) 1 and DLL4 in the hematopoietic microenvironment, and show that elevated levels of circulating vascular endothelial growth factor (VEGF) characteristic for cancer patients is one of the mediators of this effect. Remarkably, selective activation of DLL1-Notch signaling alone by over-expression of DLL1 in bone marrow precursors or by soluble multivalent DLL1 treatment significantly enhances tumor-specific immune responses and inhibits tumor growth in multiple tumor models. The data suggest that tumor growth suppresses T cell differentiation and function via inhibition of DLL-mediated Notch signaling in the hematopoietic compartment. Our findings uncover a novel molecular mechanism of immune suppression and suggest a pharmacologic intervention based on activation of DLL1-Notch signaling to reverse this effect, stimulate immune responses and suppress tumor growth. Given that recently DLL1 was also implicated in abnormal tumor vessel formation and growth inhibition [Zhang JP, ea. Cancer Lett. 2011;309:220–227], systemic stimulation of Notch by multivalent DLL1 might represent an efficient therapeutic strategy targeting multiple mechanisms of tumor growth.

Abstract B93: Resuscitating cancer immunosurveillance by combining Notch 1 and death receptor-activating therapy.

Notch signaling plays diverse roles in lymphocyte differentiation and tumor angiogenesis. Our recent findings implicate the role of the Notch 1 pathway in promoting antitumor immune effector functions. We explored the effects of Notch 1 stimulation on T cell immunity and its potential pharmacological activation using multivalent Delta-like Notch 1 ligand DLL1 cluster in combination with the proteasome inhibitor bortezomib, which could stabilize Notch 1 activation. Bortezomib was found to selectively sensitize solid tumor cells to apoptosis by upregulating death receptors on tumor cells and amplifying caspase-8 activation in the death-inducing signaling complex. This resulted in reduced pulmonary nodules of injected tumor cells in mice. Additionally, in an in vivo tumor setting, no adverse effects of bortezomib were observed on the antigen-specific T cell proliferative or cytolytic functions. But no long-term survival benefits could be obtained, possibly due to the immunosuppressive effects of the tumor. In our more recent studies, we observed that DLL1-specific immune Notch activation could enhance antitumor T cell immunity by overcoming tumor-associated immunosuppression in D459 and Lewis lung carcinoma models. The results offer a novel combinatorial approach to abrogate the immunosuppressive circuitries operating in the tumor microenvironment with a potential to reduce tumor burden and resuscitate tumor-specific immunity. This work has important translational implications for improving immunotherapy in cancer patients, and may provide insights into new strategies for cancer prevention. Citation Format: Duafalia F. Dudimah, Roman V. Uzhachenko, Samuel T. Pellom, Jr., Asel K. Biktasova, Mikhail M. Dikov, David P. Carbone, Anil Shanker. Resuscitating cancer immunosurveillance by combining Notch 1 and death receptor-activating therapy. [abstract]. In: Proceedings of the Fifth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2012 Oct 27-30; San Diego, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2012;21(10 Suppl):Abstract nr B93.