Marcelo G. Bonini - US grants

DESCRIPTION (provided by applicant): The overall objective of this research effort is to identify the causes and consequences of nitric oxide and oxidant production imbalances in health and disease, investigate mechanisms of microbial killing by the immune system, study metal homeostasis in cells and how it contributes to cellular proliferation and migration and establish the relevance of redox processes in the differentiation of stem cells. It is currently well-established that oxidant homeostasis and electron transfer reactions are fundamental in maintaining physiological processes in health and that their impairment or dysfunction cause severe phenotypical consequences. Both reduced oxidant bioavailability or overproduction and difficulties in mediating key electron transfer reactions are key factors leading to the impairment of beneficial signal transduction events or inducers of cellular and tissue damage. Oxidant imbalances are becoming recognized contributors of hypertension and cardiovascular complications in diabetes, atherosclerosis, inflammation, male impotence, and important factors contributing to stem cell differentiation and regeneration and cancer onset, progression and prognosis. All of these are human conditions of public health interest. The University of Illinois at Chicago Colleges of Medicine, Dentistry, Pharmacy and Biological Sciences through its diverse departments and research initiatives is currently developing multiple studies and programs dedicated to the understanding of how paramagnetic species that can be tracked, identified and quantified through the use of EPR lead to increased lung permeability in inflammation and sepsis, oxidative stress induced alterations of ventricular function, oxidant mediated vascular remodeling and damage, stem cell differentiation, protein interactions, cancer biology, photosynthesis, microbial killing and drug metabolism. The development of such initiatives depend, at least in part, on our ability to identify which among the many reactive oxygen and nitrogen species are produced at different stages of disease progression, and the precise quantification of reactive species yield, on our capacity to define electron donors and acceptors and track molecular events influencing membrane permeability and interaction with drugs. Electron paramagnetic resonance (EPR) remains the only available technique capable of unequivocally identifying particular paramagnetic species (stable and short lived) based on fingerprint signature resonance spectra and serves the purpose of quantifying the generation of free radical oxidants. For example, it is of pivotal importance to directly identify the sources of superoxide radical anion and nitric oxide that contribute to tissue damage in inflammation and trigger signaling events that contribute to pulmonary edema, hypertension and cardiac failure or trigger events such as stem cell differentiation. At the same time it would be possible to determine how superoxide dismutase enzymes contribute to avoid or promote oxidative stress events warranting or compromising nitric oxide bioavailability. Through spin labeling it would be possible to study membrane microdomain formation and track drug metabolites. Therefore, the acquisition of an EPR instrument would integrate and uniquely contribute for the development of the current initiatives while fostering new collaborative studies putting the departments in the position of making important progress towards the understanding of intricate molecular processes and the development of therapies to re-equilibrate physiological processes at the cellular level.

DESCRIPTION (provided by applicant): Poor prognosis of severe sepsis is associated with impaired microcirculation, multiple organ injury and disseminated intravascular coagulation (DIC) as a result of interdependent mechanisms of systemic intravascular inflammation, vascular leakage, microvascular thrombosis, and thrombocytopenia. Currently, no drug is available to concomitantly treat these events in sepsis. We recently discovered an important role for VWF/GPIb-IX- and integrin-dependent platelet adhesion and thrombus formation in sepsis (Yin ATVB 2013), and that G?/13 family of heterotrimeric G proteins play critical roles in integrin outside-in signaling in platelets (Gong et al, Science 2010, Shen et al Nature 2013) and in vWF secretion by endothelial cells (Rusu et al Blood 2014). G?/13 also negatively regulates cadherin function (Meigs JBC 2002) and increases endothelial permeability. In addition, we have identified NOS1-dependent regulation of Suppressor of Cytokine Signaling (SOCS1) expression in monocytes/macrophages (Baig et al, Science Signaling, in revision) as a critical determinant of sepsis-induced vascular inflammation and hyperpermeability. Here, we will test the hypotheses that sepsis can be effectively treated by concomitant anti-thrombotic, anti-inflammatory and anti- hyperpermeability therapy with novel agents that do not cause hemorrhage and vascular leakage. We have generated novel inhibitory peptides of G?/?AP interaction that inhibit endothelial VWF secretion as well as inhibitors of G?-integrin and vWF-GPIb-IX interactions that potently inhibit thrombosis without adversely inducing hemorrhage (Shen et al, Nature 2013; Yin et al, ATVB 2013; Rusu et al., Blood 2013). We propose to (1) determine the therapeutic effect of inhibiting GPIb and G?/13/integrin signaling in sepsis; (2) determine the role of G?-dependent endothelial cell VWF secretion in sepsis-induced microthrombosis; and (3) interfere with the inflammatory response that induces endothelial hyperpermeability in sepsis concomitantly with anti-thrombotic therapy. This study will not only provide novel therapeutic concepts in sepsis, but also translate these concepts into new anti-sepsis drugs and treatments.

DESCRIPTION (provided by applicant): Poor prognosis of severe sepsis is associated with impaired microcirculation, multiple organ injury and disseminated intravascular coagulation (DIC) as a result of interdependent mechanisms of systemic intravascular inflammation, vascular leakage, microvascular thrombosis, and thrombocytopenia. Currently, no drug is available to concomitantly treat these events in sepsis. We recently discovered an important role for VWF/GPIb-IX- and integrin-dependent platelet adhesion and thrombus formation in sepsis (Yin ATVB 2013), and that G?/13 family of heterotrimeric G proteins play critical roles in integrin outside-in signaling in platelets (Gong et al, Science 2010, Shen et al Nature 2013) and in vWF secretion by endothelial cells (Rusu et al Blood 2014). G?/13 also negatively regulates cadherin function (Meigs JBC 2002) and increases endothelial permeability. In addition, we have identified NOS1-dependent regulation of Suppressor of Cytokine Signaling (SOCS1) expression in monocytes/macrophages (Baig et al, Science Signaling, in revision) as a critical determinant of sepsis-induced vascular inflammation and hyperpermeability. Here, we will test the hypotheses that sepsis can be effectively treated by concomitant anti-thrombotic, anti-inflammatory and anti- hyperpermeability therapy with novel agents that do not cause hemorrhage and vascular leakage. We have generated novel inhibitory peptides of G?/?AP interaction that inhibit endothelial VWF secretion as well as inhibitors of G?-integrin and vWF-GPIb-IX interactions that potently inhibit thrombosis without adversely inducing hemorrhage (Shen et al, Nature 2013; Yin et al, ATVB 2013; Rusu et al., Blood 2013). We propose to (1) determine the therapeutic effect of inhibiting GPIb and G?/13/integrin signaling in sepsis; (2) determine the role of G?-dependent endothelial cell VWF secretion in sepsis-induced microthrombosis; and (3) interfere with the inflammatory response that induces endothelial hyperpermeability in sepsis concomitantly with anti-thrombotic therapy. This study will not only provide novel therapeutic concepts in sepsis, but also translate these concepts into new anti-sepsis drugs and treatments.

Environmental arsenic (As) is a class I human carcinogen with established roles in promoting skin, bladder, lung and kidney cancers. The role of arsenic as a breast carcinogen is less established although numerous studies have indicated that in cell cultures As promotes the specification of breast cancer cells towards phenotypes that are estrogen receptor negative as well as more lethal and challenging to treat. The molecular mechanisms involved remain unknown. Our laboratory found that As promotes alterations in the metabolism of mitochondrial reactive oxygen species (ROS) via inhibiting the tumor suppressor Sirtuin 3 which leads to the accumulation of manganese superoxide dismutase (MnSOD) in an acetylated/inactive form (MnSOD-Ac), increased reactive oxygen species (ROS) and the activation of hypoxia induced factor 2? (HIF2?). The activation of HIF2? is a well-established mechanism of stem cell reprogramming that has also been implicated in metastatic recurrence, as well as treatment failure in women with breast cancer. Hence, we propose that chronic As exposure is a risk factor for the development of ER(-) breast cancer via a mechanism that involves MnSOD acetylation and mitochondrial ROS. By extension, we propose that the MnSOD-Ac/HIF2? molecular signature may identify women with breast cancer that have been exposed to As and who may require personalized care for they are at increased risk of failing standard therapeutics. Also, that the MnSOD-Ac/HIF2? may be targeted to improve therapy in these women. Our aims are as follows: (1) determine if MnSOD-Ac reprograms tumor cell to stem-like (more aggressive) phenotypes associated with chemoresistance and if targeting MnSOD-Ac reverses this effect. (2) determine if low level arsenic exposure in the drinking water transforms ER+ in situ xenograph tumors developing in mice towards more pervasive phenotypes. (3) determine if there is an association between arsenic exposure and more aggressive subtypes of breast cancer with a MnSOD-Ac, or MnSOD-ROS-HIF2? molecular signature as well as if arsenic exposure promotes chemoresistance or a prevalence of aggressive ER(-) phenotypes.

The current project is focused on the study of basic mechanisms of polarization of the innate immune response. Macrophages are the primary cell type responsible for the polarization of the innate immunity either by initiating and propagating inflammation (M1 phenotype) or resolving inflammation to promote tissue healing (M2 phenotype). A relative new idea is that the dysregulation of macrophage polarization with the prevalence of M1 over M2 phenotype promotes wide-spread inflammation and acute tissue injury. In the case of lungs acute lung injury (ALI) may evolve to acute respiratory distress syndrome (ARDS), a highly lethal form of respiratory failure. Hence, understanding the mechanisms by which macrophages polarize into functionally distinct phenotypes may lead to new therapeutic strategies to prevent or treat ARDS. Nuclear factor ?B (NF?B) is a master regulator of inflammation being responsible for the expression of genes that promote or resolve inflammation. While the p65/p50 configuration of NF?B promotes inflammation, the p50/p50 configuration promotes resolution. We reason that these configurations also regulate how macrophages polarize into distinct phenotypes. In this regard, we recently discovered that suppressor of cytokine signaling-1 (SOCS1) may operate the switch in NF?B function since it targets nuclear p65 but not p50 to degradation changing the relative abundances of these component subunits in the nucleus. The finding that SOCS1 is sensitive to inhibition by nitric oxide (NO) and possibly other reactive species also indicate novel molecular mechanisms by which SOCS1 activity, NF?B function, pro- and anti-inflammatory transcription, as well as macrophage polarization and inflammatory outcomes are regulated. These mechanisms are the focus of our three proposed aims: (1) to determine the mechanism by which SOCS1 promotes NF?B functional switch from that of a pro- to that of an anti-inflammatory transcriptional complex; (2) to determine if changes in the intracellular redox state of macrophages affects SOCS1 activity and NF?B function as a transcription factor and (3) Determine how SOCS1 expression in different immune cell types regulates the transition between inflammation propagation and resolution in a mouse model of bacterial pneumonia.

ABSTRACT Environmental inorganic arsenic (iAs) is a class I human carcinogen with established roles in promoting skin, colon, bladder and kidney cancers. The role of iAs as a breast carcinogen is less established although numerous studies have indicated that in cell cultures iAs promotes the specification of breast cancer cells towards phenotypes that are estrogen receptor negative which are more lethal as well as more challenging to treat. The molecular mechanisms involved remain unknown. Our laboratory found that iAs promotes alterations in the metabolism of mitochondrial reactive oxygen species (ROS) via inhibiting the tumor suppressor Sirtuin 3 which leads to the accumulation of manganese superoxide dismutase (MnSOD) in an acetylated form (MnSOD-Ac), increased reactive oxygen species (ROS) and the activation of hypoxia induced factor 2? (HIF2?). The activation of HIF2? is a well-established mechanism of stem cell reprogramming that has also been implicated in metastatic recurrence as well as treatment failure in women with breast cancer. Hence, we propose that chronic iAs exposure is a risk factor for the development of ER(-) breast cancer via a mechanism that involves MnSOD acetylation and mitochondrial ROS. By extension, we propose that the MnSOD-Ac/HIF2? molecular signature may identify women with breast cancer that have been exposed to iAs and required personalized care for they are at increased risk of failing standard therapeutics. Also, that the MnSOD-Ac/HIF2? may be targeted to improve therapy in these women. Our aims are as follows: (1) determine if MnSOD-Ac reprograms tumor cell to stem-like (more aggressive) phenotypes associated with chemoresistance and if targeting MnSOD-Ac reverses this effect. (2) determine if low level iAs exposure in the drinking water transforms ER+ in situ xenograph tumors developing in mice towards more pervasive phenotypes. (3) determine if there is an association between exposure to iAs and breast cancer with a MnSOD-Ac, or MnSOD-ROS- HIF2? molecular signature as well as if iAs exposure promotes chemoresistance or a prevalence of aggressive ER(-) phenotypes.

PROJECT SUMMARY Aggressive phenotypes of breast cancer are characterized by increased capacity of evading immune surveillance and generating metastasis. Our groups recently described that metastatic breast cancer cells expressing high levels of acetylated superoxide dismutase 2 (SOD2K68Ac) accumulate mitochondria-derived reactive oxygen species (mtROS) and promote stabilization of hypoxia-inducible factor 2? (HIF2?), involved tumor aggressiveness. HIF2? also regulates genes associated with immune protection, including the programmed death-ligand 1 (PDL1) that is widely recognized as a molecule eliciting immune evasion in cancer cells. Hence, we propose to investigate (1) if accumulation of SOD2K68Ac promotes PDL1 upregulation via HIF2? in breast cancer cells; (2) if SOD2K68Ac/HIF2? promotes mammary cancer immune evasion and metastasis in vivo using mouse model; and (3) if there is a subgroup of women with breast cancer that exhibits SOD2K68Ac/HIF2? molecular signature correlating with high PDL1 expression and immunotherapy resistance. We expect to identify a new mechanism of cancer immune evasion that can be targeted to improve therapeutic approaches to treat aggressive phenotypes of breast cancer exhibiting SOD2K68Ac molecular signature. The proposed translational research will be conducted by Dr. Coelho under the mentorship of Dr. Marcelo Bonini (primary mentor), Dr. Leonidas Platanias (co-mentor) and Dr. Massimo Cristofanilli (clinical co-mentor). All mentors have exemplary records of scientific achievement, innovation and leadership in the translational pipeline of cancer therapeutics and diagnostics. In addition to the scientific goal of this application, we expect that Dr. Coelho will accomplish a multidisciplinary training in clinical research, leadership, writing and management essential to establish an independent career in cancer immunobiology.

ABSTRACT ? CANCER RESEARCH CAREER ENHANCEMENT AND RELATED ACTIVITIES The Lurie Cancer Center (LCC) provides a comprehensive training environment to enhance the career development of cancer researchers and caregivers at all levels. Education and training at the LCC is led by Dr. John Crispino, Associate Director for Education and Training since 2012, with support of several faculty and LCC staff. The combination of substantial NIH training grant funding and dedicated philanthropic support enables numerous training opportunities for students, research fellows, clinical fellows, oncology nurses, and junior faculty. These include numerous seminar series, symposia, retreats, intramural grants, and travel awards. The LCC manages five NCI T32 grants, and trainees are further supported by four other cancer relevant T32 grants and two K12 grants. New to this cycle are a formal mentorship program for clinical trainees and the Translational Bridge Initiative, which provides combined laboratory and clinical mentor oversight for selected post-doctoral fellows. In addition, there are LCC-sponsored education opportunities for health care professionals and oncologists in the Chicago area, nationally and internationally. Over the next funding period the LCC will continue to expand the ongoing activities, optimize training for precision medicine and other emerging fields and intensify efforts to increase the diversity of our trainees.