Shigeki Miyamoto - US grants

Cancer often develops through the disruption of proper responses to extracellular signals. Thus, understanding how normal cells sense and generate proper responses to changes in environment is important. Normally, transduction of extracellular signals into cellular responses involves a cascade of biochemical events that eventually induce gene expression. These events are mediated by transcription factors (TF), a class of DNA-binding proteins which dictate the nature of genes expressed. Deregulation of TF activities can lead to amplification of anomalies via uncontrolled gene expression. Activation of a critical TF, NF-kappaB, by extracellular signals normally occurs only transiently since NF-kappaB activates synthesis of its own inhibitor, IkappaBalpha. By contrast, deregulation of NF-kappaB activity, frequently seen in human cancers, must counteract this feedback mechanism to maintain constitutive (constant) NF-kappaB activation. How and why is NF-kappaB anomalously activated? Research in this laboratory has demonstrated that murine B cells, a rare example with non-pathological constitutive NF- kappaB activation, maintain such activity by degrading newly synthesized IkappaBalpha via a previously uncharacterized mechanism. Our preliminary data also suggest that certain human breast cancer cells aberrantly activate NF-kappaB by utilizing this alternative mechanism. Thus, the proposed research will test the hypothesis that a novel IkappaBalpha degradation mechanism is required for constitutive NF- kappaB activation in B cells and certain human breast cancer cells. In aim 1, mutational analysis will be employed to identify IkappaBalpha sequences that specify degradation by this novel pathway. Under Aim 2, the functional role of this alternative pathway will be determined in both induction and maintenance phases of constitutive NF-kappaB activation in B cells, by further specifying how proteasome-dependent NF-kappaB activation becomes proteasome-independent via a switch in IkappaBalpha degradation mechanisms. Aim 3 will test the mechanism and role of constitutive NF-kappaB activation in breast cancer cells by focusing on the alternative IkappaBalpha degradation in relation to cell survival function. This research program will provide not only a mechanistic insight into how an alternative pathway restricted to normal B cell function may be abnormally employed by cancer cells by also molecular reagent tools to probe other constitutive NF-kappaB activation systems.

DESCRIPTION (As Adapted From the Investigator's Abstract): DNA damaging agents are an important component of anti-cancer therapy. These agents elicit numerous cellular responses, including the induction of apoptosis. Recent data suggest that the NF-kappaB transcription factor activation may have anti-apoptotic effects. Understanding the mechanisms of NF-kappaB activation may lead to improved tumor killing by attenuating downstream antiapoptotic. However, the exact mechanism of NF-kappaB activation after specific DNA damage is unknown. This proposal focuses on the mechanisms of NF-kappaB activation by camptothecin (CPT-related anticancer agents, which are Topoisomerase (Topo) I inhibitors. Inactive NF-kappaB is present in the cytoplasm and its activation requires nuclear translocation. How is cytoplasmic NF-kappaB activated by nuclear DNA damage? Studies in this laboratory strongly suggest the presence of a nuclear to cytoplasmic signaling pathway. Three Specific Aims are proposed to test the hypothesis that Topo-I generated DNA double-stranded breaks in the nucleus initiate a signaling pathway that ultimately activates IkappaB kinase to release NF-kappaB from its inhibitor IkappaB. Two aims will specify the nuclear signaling events triggered by CPT treatment by focusing on the role of DSB (Aim I) and DNA-dependent protein kinase (DNA-PK, Aim 2). Cell cycle expression of an NF-kappaB-responsive reporter and restriction enzyme mediated generation of DSB will determine whether DSB is sufficient for NF-kappaB activation. The critical involvement of DNA-PK will be uncovered by assaying the NF-kappaB activity in genetically matched cells from mice deficient for each of the kinase complex components. Finally the endpoint of the signaling in the cytoplasm will be specified by focusing on the CPT-dependent regulation of an IkappaB kinase complex that phosphorylates the inhibitor IkappaB to ultimately release active NF-kappaB (Aim 3). Aim I will determine the target specificity of Topo-I-mediated DNA damage to trigger NF-kappaB activation.

DESCRIPTION (provided by applicant): Cancer often develops through the disruption of proper responses to extracellular signals. Thus, understanding how normal cells sense and generate proper responses to changes in environment is important. Normally, transduction of extracelluar signals into cellular responses involves a cascade of biochemical events that eventually induce gene expression. These events are mediated by transcription factors (TF), a class of DNA-binding proteins that dictate the nature of genes expressed. Deregulation of TF activities by genetic and epigenetic anomalies can lead to amplification of disrupted cellular responses via uncontrolled gene expression. Activation of a critical TF, NF-kappaB, by extracellular signals normally occurs only transiently since NF-kappaB activates synthesis of its own inhibitor, IkappaBalpha, which enters the nucleus, removes NF-kappaB from DNA binding sites, and exports it out to the cytoplasm to terminate NF-kappaB function. By contrast, deregulation of NF-kappaB activity, frequently seen in human cancers, must counteract this feedback mechanism to maintain constitutive (constant) NF-kappaB activation to sustain survival and induce chemo/radioresistance. Research in this laboratory has demonstrated that murine B cells, a rare example with non-pathological constitutive NF-kappaB activation, maintain such activity by degrading newly synthesized IkappaBalpha via a previously uncharacterized mechanism. Our preliminary data also suggest that in order for continual degradation of IkappaBalpha to occur, there is a requirement that newly formed, nuclear NF-kappaB/IkappaBalpha complexes must be exported out to the cytoplasm in both murine B cells and human cancer cells. Thus, the proposed research will test the hypothesis that constitutive NF-kappaB activation requires a mechanism to counteract the autoinhibitorv feedback regulation imposed by its inhibitor IkappaBalpha. In Aim 1, mutational analysis will be employed to delineate novel IkappaBalpha degradation mechanisms. Under Aim 2, the functional role of the nuclear export of IkappaBalpha will be determined in both maintenance of constitutive NF-kappaB activation and survival of human cancer cells. Aim 3 will test the in vivo roles of nuclear export of IkappaBalpha in B cell development by the generation of mice harboring IkappaBalpha loci with N-NES point mutations. This research program will help define fundamental mechanisms critical for constitutive NF-kappaB activation in B cell development and human malignancies. They may also reveal N-NES-mediated nuclear export of IkappaBalpha as a rational therapeutic target to generally disrupt constitutive NF-kappaB activation to induce cell death or chemo/radiosensitization in human cancer.

DESCRIPTION (provided by applicant): Activation of the transcription factor NF-KB by DNA-damaging anticancer agents has emerged as an important modulator of malignant behaviors, including resistance to apoptotic cell death. This signal transduction pathway also serves as an attractive paradigm to understand how nuclear DNA damage induces a signaling pathway to activate a cytoplasmically sequestered transcription factor - a nuclear-to-cytoplasmic signal transduction pathway. The long-term goal of this proposal is to increase our understanding of the mechanisms involved in NF-KB signal transduction pathways initiated by the model DNA-damaging anticancer agents, camptothecin and etoposide. In our published studies, we provided evidence that a convergence of two parallel pathways is induced by these genotoxic agents: (a) DSB-dependent activation of ATM; and (b) a stress signal that leads to nuclear accumulation of NEMO/IKKy, the regulatory subunit of the kB kinase (IKK) complex, via a SUMO-1 modification. Ultimately, NEMO exports in an ATM- and ubiquitin-dependent manner and activates the cytoplasmic IKK complex. Thus, NEMO represents a common nuclear signal that mediates the nuclear-to-cytoplasmic signaling pathway induced by these distinct anticancer agents. Through the following Specific Aims, we will test the hypothesis that a series of NEMO posttranslational modifications mediates its nuclear import and export to chaperone an IKK regulator from the nucleus to the cytoplasm to promote IKK activation in response to genotoxic stimuli. Aim 1: Specify the mechanism of SUMO-1 modification of NEMO by genotoxic agents. Aim 2: Determine the role of ubiquitin in nuclear export of NEMO in genotoxic signaling. Aim 3: Elucidate the role of ATM in IKK activation by genotoxic agents. The proposed studies will help determine the mechanisms fundamental to NF-KB activation by certain genotoxic anticancer agents. These mechanisms may also serve as models for activation pathways induced by other genotoxic agents, such as ionizing radiation, and other agents that target DNA topoisomerase I or II. Moreover, new reagent tools, such as phospho-specific NEMO antibodies, cDNAs encoding wild type and mutant versions of SUMO and ubiquitin ligases for NEMO, and cell systems that harbor these mutant proteins, will also be useful for other investigators in their efforts to dissect NF-KB activation pathways. Finally, specific enzymes and processes involved in this activation pathway may serve as rational therapeutic targets to induce chemo/radio-sensitization in certain types of human cancer.

Constitutive NF-[unreadable]B activation-persistent activation in the absence of added stimuli-is observed in developing B lymphocytes and in the pathological setting of human cancer. This activity is important for maturation of B cells and survival and chemo/radioresistance of cancer cells. Because NF-[unreadable]B can be activated by a wide variety of distinct mechanisms, constitutive activation may involve complex, deregulated mechanisms in different cancer settings. The long- term goal of this proposal is to increase our understanding of fundamental mechanisms controlling constitutive NF-[unreadable]B activity. There are five NF-[unreadable]B/Rel transcription factors that function as dimers. Two major NF-[unreadable]B complexes are p65:p50 and cRel:p50 heterodimers. Depending on cellular context, one or both of these can be constitutively activated. A unique aspect of this system is that typically activation of these complexes occurs transiently through a feedback loop with its inhibitor, I[unreadable]B[unreadable], which exports NF-[unreadable]B to the cytoplasm to terminate activity. Constitutive NF-[unreadable]B activation thus requires a mechanism to overcome this intrinsic negative feedback regulation and sustain high activity. Based on research during the previous funding period, we hypothesize that p65 and cRel have unique intrinsic mechanisms that establish different constitutive activation threshold involving unique (i) nuclear export mechanisms and (ii) I[unreadable]B[unreadable]-binding affinities. Moreover, these mechanisms are further enhanced in certain cases by (iii) constitutive degradation of newly synthesized I[unreadable]B[unreadable] via a nonclassical pathway that we identified. Specific aims are: 1. To determine the role of nuclear export in regulating constitutive NF-[unreadable]B activation. 2. To reveal the role of I[unreadable]B[unreadable]-binding affinity in specifying constitutive NF-[unreadable]B activation. 3. To further delineate constitutive NF-[unreadable]B activation mechanisms via nonclassical I[unreadable]B[unreadable] degradation. These proposed studies will increase our understanding of fundamental mechanisms controlling specificity and threshold of constitutive activation of different NF-[unreadable]B complexes, which may help identify key mechanisms to induce chemo- and/or radiosensitization in human cancer, regardless of upstream deregulated pathways involved. Finally, they will also generate novel tools that can be used to analyze critical tumor microenvironment components in promoting constitutive NF-[unreadable]B activity in different human malignancies.

DESCRIPTION (provided by applicant): The protein kinase Akt provides strong survival signals in cardiomyocytes, both chronically through altered gene programming, and more acutely through mechanisms that have not been fully elucidated. This proposal examines the hypothesis that activated Akt translocates to mitochondria and provides acute cardioprotection via its ability to phosphorylate hexokinase-II (HK-II) and enhance HK-II association with and inhibition of permeability transition pore (PT-pore) opening. Data I have generated in my published work show that Akt translocates to mitochondria upon acute activation by leukemia inhibitory factor (LIF), phosphorylates HK-II and prevents H202 and Ca2+induced PT-pore opening in neonatal rat ventricular myocytes or mitochondria isolated from adult mouse heart. Protective responses mediated by Akt are impaired by dissociation of HK-II. Aim #1 of this proposal seeks to establish a requirement for mitochondrial HK-II and its phosphorylation in Akt-mediated protection against PT-pore opening. This will be tested using loss and gain of function approaches in which mitochondrial HK-II is decreased by an HK-II dissociating peptide, and increased by adenoviral expression of WT HK-II, a mutated HK-II lacking its mitochondrial binding motif or kinase dead HK-II. The requirement for HK-II phosphorylation will be tested by using HK-II mutated to either prevent or mimic phosphorylation at its putative Akt phosphorylation site. To extend these findings to the adult heart, Aim #2 will examine regulation of mitochondrial Akt and HK-II and the phosphorylation of HK-II using Langendorff perfused heart model and in vivo. The effects of HK-II dissociation from mitochondria and of TAT-fusion HK-II and mutants into the heart will be tested for their effects on ischemia/reperfusion (I/R) damage in perfused heart in the presence or absence of IGF-1 or in vivo. In Aim #3, I will determine whether the recently discovered Akt-phosphatase, PHLPP-1, localizes to mitochondria and regulates mitochondrial Akt activity and thus mitochondrial HK-II/PT-pore opening. This will be examined by knockdown of PHLPP (using siRNA and PHLPP knock-out mice) and by increasing PHLPP-1 expression via adenoviral expression. These experiments will provide evidence for HK-II as a potential downstream target of Akt, and PHLPP as a potential regulator of Akt, and will suggest novel sites of intervention for inhibiting I/R induced mitochondria mediated cardiomyocyte cell death. PUBLIC HEALTH RELEVANCE: Cardiomyocyte death plays a crucial role in heart disease and opening of the permeability transition pore (PT-pore) in mitochondria is a major executor of cell death. The protein kinase Akt provides strong survival signals in cardiomyocytes and the mechanisms by which Akt prevents cell death have not been fully elucidated. This proposal examines the hypotheses that activated Akt translocates to mitochondria and provides acute cardioprotection via its ability to phosphorylate hexokinase-II (HK-II) and that this protective signaling is regulated by a Akt phosphatase at mitochondria.

DESCRIPTION (provided by applicant): Activation of NF-kB by DNA-damaging anticancer agents, including ionizing radiation (IR), has emerged as an important modulator of malignant cell behaviors, such as resistance to apoptotic cell death. This signal transduction pathway also serves as a paradigm to understand how nuclear DNA damage may induce nucleus-to-cytoplasmic signal transduction pathways. We have previously discovered a novel nucleus-to-cytoplasmic NF-kB signaling pathway induced by IR and other agents that can induce DNA double strand breaks (DSBs). This signaling pathway involves a post-translational modification (PTM) of NEMO (NF-kB essential modulator)/IKK3, the regulatory subunit of the I:B kinase (IKK) complex, by SUMO-1 (small ubiquitin-like modifier 1). We now generated a novel NemoDK knockin mice harboring a germ-line mutation of the sumoylation sites. In the current proposal, we will establish the physiological importance of this new NF-kB signaling pathway in response to IR by directly evaluating the role of NEMO sumoylation in vivo and further dissect the critical upstream and downstream mechanisms. The following three aims will address our central hypothesis that NEMO sumoylation plays a critical physiological role in mediating NF-kB activation by IR to modulate radiation sensitivity in vivo: Aim 1: Determine the roles of NEMO sumoylation in modulating radiation sensitivity in vivo. Aim 2: Reveal the upstream role of NEMO zinc finger in promoting NEMO sumoylation. Aim 3. Elucidate SUMO-1 specific downstream regulation of NEMO function. We believe that the proposed research is important for two major reasons. First, the described research will uncover novel NF-kB signal transduction mechanisms in response to DNA damage stimuli. Second, understanding the mechanisms of NF-kB activation by DNA damaging agents may help identify novel drug targets to improve the current methods of cancer therapy. PUBLIC HEALTH RELEVANCE: Many anticancer agents, including ionizing radiation (IR), are used to kill cancer cells, but unfortunately they also turn on cancer cell survival mechanisms in cancer cells that counter the death effect. One of these mechanisms is the activation of the transcription factor NF-kB that rapidly turns on the synthesis of survival genes. The proposed research will uncover important mechanisms involved in this activation, which will help identify novel drug targets to improve the current methods of cancer therapy.

DESCRIPTION (provided by applicant): The NF-?B/Rel family of transcription factors contributes to critical cellular processes, including immune, inflammatory and cell survival responses. As such, NF-?B is implicated in immunity-related diseases, such as autoimmunity, as well as multiple types of human malignancies. Understanding mechanisms of NF-?B regulation will not only expand our knowledge of basic cell signaling processes but also provide potential avenues to prevent and/or treat these human disorders. While a large body of literature over the last two decades describes the critical roles of ubiquitin in regulating NF-?B functions, very little is known about regulation of NF-?B signaling by SUMO (small ubiquitin-like modifier), another posttranslational modifier. The long-term goal of this project is to greatly expand our understanding of the mechanisms of NF-?B and SUMO regulation in specific physiological and pathological processes. We have recently uncovered a novel signaling role for SUMOylation of NEMO (NF-?B essential modulator) in NF-?B signaling. Our preliminary data indicate that there exist significant, novel crosstalk mechanisms between the SUMO and NF-?B pathways. Thus, in this proposal, we will test the hypothesis that crosstalk between SUMO and NF-?B signaling systems plays critical roles in regulating certain physiological and pathological processes. This research is expected to considerably expand our knowledge of the molecular links between SUMO and NF-?B pathways and their roles in specific physiological and pathological processes. This research will also generate novel reagents and tools to allow other researchers to investigate SUMO and NF-?B signaling systems in similar and different experimental models. Finally, it may also identify rational targets for drug development against human disorders, such as autoimmunity and specific types of malignancies. PUBLIC HEALTH RELEVANCE: The regulation of cancer cell death is a complex process involving many different molecular pathways. This research seeks to understand the relationships between protein modification by SUMO (Small Ubiquitin-like Modifier) and NF-?B signaling, one of the major cell death-regulatory pathways. This study will significantly expand our understanding of the regulatory mechanisms for normal and cancer cell death signaling, and may also provide rationale targets for the development of new anticancer drugs.

DESCRIPTION (provided by applicant): In contrast to highly sensitive genomic and proteomic methods that primarily evaluate the state of cancer specimens, functional analyses of primary patient samples to assess their biological responses to various experimental conditions are difficult because of the often limited number of live primary cells that can be obtained from patient biopsy samples. Thus, overcoming this technical barrier has the potential to transform our ability to significantly increase translational cancer research approaches. By combining expertise in, cancer cell signaling, bioengineering, and primary patient care, the Miyamoto-Beebe-Callander team proposes to improve our ability to functionally analyze NF-kB signal transduction responses in primary patient multiple myeloma (MM) samples. Specifically, we propose to develop innovative microchannel culture devices and implement functional studies to investigate a dogma-challenging NF-kB-survival pathway in MM. We have already developed prototype culture devices that increase our ability to analyze primary MM cells. These microculture systems also provide the flexibility to study components of the tumor microenvironment, such as tumor-supporting bone marrow stromal cells (BMSCs). Under Aim 1 we will dissect drug resistance-inducing NF-kB signaling mechanisms in primary MM cells using the first generation microscale cell culture chambers (MCCCs). We also aim to reveal patient individualized information by co- culturing MM cells and BMSCs derived from the same patients, a paradigm shift from the conventional experimental setup where patient sources of MM and BMSCs are randomly mixed. Under Aim 2 we will further improve functional micro-scale assays with additional functionalities and with even smaller cell numbers per condition, thus greatly expanding the scope of translational research in MM. The micro-culture technology proposed has the potential to rapidly change the methods used for investigating signal transduction studies, including NF-kB, in MM and other blood and possibly solid cancer types.

DESCRIPTION (provided by applicant): The heart is a high-energy demand organ and ischemic heart disease is a significant cause of morbidity and mortality. During ischemia, macroautophagy (hereafter referred to as autophagy) is induced to sequester intracellular contents and organelles including mitochondria for lysosomal degradation resulting in preservation of cellular energy status and conferring cell survival. Mitochondrial autophagy (mitophagy) is a selective type of autophagy which also plays a protective role by eliminating compromised mitochondria, thereby limiting the activation of mitochondrial cell death pathways as well as supplying an energy source. Hexokinase-II (HK-II) catalyzes the first step of glycolysis, phosphorylating glucose, and is the predominant isoform in the heart. HK-II is increasingly recognized as a survival signaling nexus, especially as a protective molecule at mitochondria against acute oxidative stress such as reperfusion injury. Building on our recently published and preliminary data, this study will examine a new facet of HK-II survival signaling: the coordination of metabolic status with mitophagy and autophagy to enhance cell survival during metabolic suppression. Aim 1 will determine whether dissociation mitochondrial HK-II (mitoHK-II) plays a regulatory role in recognition of damaged mitochondria to limit cardiac damage induced by ischemia in cardiomyocytes using adenoviral expression of mitoHK-II dissociating peptide (15NG). We will determine whether HK-II binds to mitofusin-2, Parkin receptors at mitochondria to regulate mitophagy. In Aim 2, we will determine whether HK-II regulates non-selective autophagy during ischemia in cardiomyocytes. Based on our recent study, we hypothesize that HK-II binds to and inhibits mTOR complex 1 (TORC1) to increase protective autophagy under ischemia and that this binding occurs at lysosome, a TORC1 activation site, in response to energy depletion. In Aim 3, we will determine whether mitophagic and autophagic effects of HK- II emerged during ischemia play a protective role against ischemic stress in vivo heart using 15NG transgenic mice generated in the lab as well as in vivo expression of HK-II mutants using adeno-associated virus serotype 9 (AAV9) technique. The long term goal of this proposal is to unveil a previously unrecognized link between HK-II and protective autophagy as a potential target for therapeutic intervention in heart diseases.

? DESCRIPTION (provided by applicant): Nuclear factor kappa B (NF-?B) is a group of dimeric transcription factors that normally regulate immune, inflammatory and cell death responses and is often deregulated in various human cancer cell types. A large body of work over the last two decades has characterized two genetically and biochemically distinct pathways of NF-?B activation, referred to as the canonical and non-canonical pathways. Common to both NF-?B activation pathways is their reliance on 26S proteasomes to degrade/process inhibitor proteins; therefore, these pathways are highly sensitive to proteasome inhibitors, including clinically employed bortezomib. Our lab previously described an atypical mechanism of NF-?B activation, which contrasted with these known pathways by its high resistance to over 10 different proteasome inhibitors, including bortezomib. We have also provided evidence that a significant fraction of NF-?B activity detectable in primary multiple myeloma (MM) and mantle cell lymphoma patient samples is resistant to bortezomib. We further reported that bone marrow stromal cells derived from MM patients', but not normal, marrows produce a soluble factor(s) that further augment NF-?B activity in MM cells to induce bortezomib resistance. We now identified one factor produced by stromal cells that can potently induce NF-?B activation in MM cells in a manner highly resistant to bortezomib. This factor is an extracellular matrix protein that has previously not been linked to NF-?B signaling or MM pathologies. Interestingly, not the full-length version but its fragments are potent inducers of NF-?B. Moreover, there are other proteins in human proteome that contain the conserved domain. Based on these new findings, we hypothesize that there are a family of novel inducers that cause atypical NF-?B activity. This hypothesis will be addressed in two specific aims: to elucidate physiologically relevant forms of this inducer and associated gene signatures (Aim1) and to determine whether other conserved domains can also induce NF-?B signaling (Aim 2). Our study may reveal a potential family of novel NF-?B inducers generated from extracellular matrix proteins that may play key roles in MM drug resistance but also other physiological and pathological processes.

UWCCC Developmental Therapeutics (DT) Program Summary Co-Leaders: Glenn Liu, Shigeki Miyamoto, and Jing Zhang PROJECT SUMMARY/ABSTRACT The mission of the UW Carbone Cancer Center (UWCCC) Developmental Therapeutics (DT) Program is to improve cancer patient outcomes by discovering new targets, developing therapeutic agents and biomarkers, and translating this research into early phase clinical trials. The DT Program provides the translational direction not only for DT members, but for other programs within the UWCCC (e.g. GEM, VR, TM). Clinical trial data and resources inform our understanding of response and resistance mechanisms and enable the identification of new therapeutic targets and treatment strategies to improve clinical outcomes. The DT Program has 59 core members, representing 18 departments and 5 different schools/colleges. Research impacts of DT members are evidenced by research awards totaling over $22.5M in annual direct cost of peer-reviewed and non-peer reviewed funding (including $3.45M NCI, $6.2M cancer-related other NIH agencies, $4.97M peer-reviewed non-NIH sources, $7.9M non-peer reviewed funding) and a significant number of publications (1228 publications, 18% of which result from intra-programmatic collaboration and 24% from inter-programmatic work). A key component of the new UWCCC Strategic Operating Plan is to further strengthen and develop Innovative Therapies and Cancer Biomarkers. Consistent with this strategic goal, the thematic aims of the DT Program are to: 1) discover new molecular targets for cancer therapy; 2) develop new agents and biomarkers; and 3) translate new therapies and biomarkers into clinical trials. To achieve this goal, it is critical to have a very strong program that discovers, develops, and translates novel interventions and biomarkers. To this end, members of the DT Program come from basic science, applied science, and clinical departments. This program has expertise ranging from basic discovery (molecular pathways and targets), to development (drugs/assays/biomarkers), to early clinical translation application (Phase 1 clinical trials). Translational and clinical research DT program members apply intra- and inter-programmatic research discoveries in clinical trials, performing the majority of first-in-human or early phase clinical trials, while individual disease-specific research teams (Disease Oriented Teams) focus primarily on phase 2 and 3 studies. DT members lead or participate in all clinical research groups facilitating both translational and reverse-translational opportunities.

ABSTRACT The heart responds to stress through hypertrophic growth of cardiomyocytes and progresses to heart failure when stress is sustained. Our previous studies showed that hypertrophy in response to a variety of stimuli occurs independent of signaling through the calcium/calmodulin dependent protein kinase II (CaMKII) but that progression to heart failure is attenuated when CaMKII is deleted. Inflammation is a key contributor to the adverse remodeling associated with heart failure. The long term objective of this proposal is to demonstrate that CaMKII signaling within cardiomyocytes initiates cardiac inflammation in response to non-ischemic interventions such as pressure overload (TAC) and that this plays a significant role in the development of heart failure. Studies proposed in Aim 1 determine if cardiomyocyte localized CaMKII signaling drives cardiac inflammation using cardiac specific CaMKII knockout mice (CKO) to demonstrate loss of TAC-induced inflammatory gene expression and inflammasome activation. We determine if these responses occur specifically in cardiomyocytes by isolation of adult mouse ventricular myocytes from CTL and CKO mice, and by in situ hybridization and enzymatic assays in tissue sections. CaMKII?C transgenics and mice with cardiac specific KO of the p65 subunit of NFkB are used to further demonstrate involvement of the cardiomyocyte in igniting inflammation. Aim 2 asks whether cardiomyocyte CaMKII signaling contributes to accumulation of inflammatory/immune cells in response to TAC. Work in this aim uses CKO mice to demonstrate that TAC promotes immune cell responses through cardiomyocyte CaMKII initiated signals. Studies focus on macrophages and T-cells, using immunohistochemistry and flow cytometry as well as single cell RNA seq to comprehensively define specific populations of macrophages that accumulate in the heart. Cardiac specific KOs or knockdown of chemokines/cytokines is used to demonstrate that generation of these mediators in cardiomyocytes triggers responses of specific immune cell types. Aim 3 determines if blockade of cardiomyocyte CaMKII-initiated inflammation attenuates adverse remodeling and at what point this needs to be accomplished. Proposed studies use cardiac specific KO or inhibition of selected inflammatory mediators to demonstrate that their formation in cardiomyocytes is critical for development of fibrosis and ventricular dysfunction following TAC. Conditional gene deletion with AAV9 Cre is used to establish the time at which maximal benefit from CaMKII inhibition is achieved. Our findings should significantly impact future research since the cardiomyocyte has not previously been considered as a generator of inflammatory signals, the mechanisms by which inflammatory responses are activated in the absence of ?alarmins? has not heretofore been determined, and the concept that most effective prevention of heart failure development could be achieved by early cardiomyocyte-targeted anti-inflammatory interventions is novel.

Multiple myeloma (MM) is the second most common hematologic malignancies and is currently considered incurable with a 5-7 year median survival. Newer drugs, such as the proteasome inhibitors, immunomodulatory drugs and monoclonal antibodies in combination with more traditional drugs enable better clinical responses. However, development of resistance to these drugs is still the major cause of patient demise. There is also a significant fraction of newly diagnosed MM patients who are refractory even to these newer drugs and thus have not benefited from the recent therapeutic advancements. We previously found that many MM patient-derived bone marrow mesenchymal stromal cells (BMSCs), a key MM tumor microenvironment cell type, secrete factor(s) capable of activating transcription factor NF-?B and causing proteasome inhibitor resistance in MM cells. We now identified HAPLN1 (hyaluronan and proteoglycan link protein 1) as a responsible BMSC secreted factor that also causes such drug resistance in MM cells in vitro and in vivo. RNA-seq and bioinformatic analyses revealed that HAPLN1 induces large-scale transcriptomic changes, including induction of a host of antiapoptotic genes. Accordingly, HAPLN1 also causes resistance to multiple other drugs in MM cells in vitro. HAPLN1 expression is higher in MM BMSCs relative to normal BMSCs, and proteolytic forms of HAPLN1 are often detected in bone marrow plasma from highly therapy refractory MM patients. Thus, we hypothesize that HAPLN1 is a new oncogenic factor and multi-drug resistance inducer in MM disease. This hypothesis will be tested by determining the pathologic role of HAPLN1 in primary MM patient cells and in vivo (Aim 1), elucidating the mechanism of HAPLN1-mediated drug resistance in MM (Aim 2), and immuno-targeting HAPLN1-mediated drug resistance in MM (Aim 3). Overall, the proposed study may identify soluble HAPLN1 as a novel marker for therapy resistance in MM, as well as a new therapeutic target to prevent or reduce the multi-drug resistance problem in MM.

PROJECT SUMMARY / ABSTRACT Investigation of cytoplasmic-to-nuclear (C?N) NF-?B signaling pathways induced by various cell surface receptors has significantly expanded the knowledge regarding the role of this transcription factor family in regulating immune/inflammatory responses and tumorigenesis. By contrast, the physiological role of DNA damage-induced nuclear-to-cytoplasmic (N?C) NF-?B signaling remains poorly understood. The proposed study will fill this knowledge gap by elucidating a surprising and crucial role of N?C NF-?B signaling in sustaining anti-tumor CD8 T cell responses during radiotherapy (RT) in vivo. The current proposal utilizes a genetically modified mouse model that selectively disables N?C NF-?B signaling in vivo. Our preliminary data show that radiation therapy can induce sustained regression of syngeneic tumors in a manner dependent on CD8 T cells. We also found that a special type of memory CD8 T cells implicated in tumor control is expanded in this mouse model. Finally, we have generated an encouraging data with an NF-kB DNA binding inhibitor to induce sustained tumor regression following radiation therapy. Based on these observations, we hypothesize that inhibition of N?C NF-?B signaling in the host improve radiation therapy via generation of tumor antigen-specific memory CD8 T cells. We will test this hypothesis by define the cellular mechanism of sustained tumor control mediated by inhibiting host N?C NF-?B signaling in radiation therapy (Aim 1), elucidate the molecular mechanism of sustained tumor control mediated by inhibiting host N?C NF-?B signaling in radiation therapy (Aim 2) and target host N?C NF-?B signaling with a chemical inhibitor to improve radiation therapy (Aim 3). The proposed study is significant because the physiological role of N?C NF-?B signaling in modulating host tumor response is completely undefined and this study will fill this knowledge gap. It is innovative because a new mouse model and a novel chemical inhibitor currently undergoing Phase 2 clinical trials will be employed. Finally, a high impact is expected because chemical targeting of N?C NF-?B signaling by the above inhibitor may expedite timely translation to clinical trials.