Browsing by Author "Pendergast, Ann Marie"

The vasculature has a crucial function in normal physiology, enabling the transport of oxygen and nutrients to cells throughout the body. In turn, endothelial cells, which form the inner-most lining of blood vessels, are key regulators of vascular function. In addition to forming a barrier which separates the circulation from underlying tissues, endothelial cells respond to diverse extracellular cues and produce a variety of biologically-active mediators in order to maintain vascular homeostasis. Disruption of normal vascular function is a prominent feature of a variety of pathological conditions. Thus, elucidating the signaling pathways regulating endothelial function is critical for understanding the role of endothelial cells in both normal physiology and pathology, as well as for potential development of therapeutic interventions.

In this dissertation, we use a combination of pharmacological inhibition and knockdown studies, along with generation of endothelial conditional knockout mice, to demonstrate an important role of the Abelson (Abl) family of non-receptor tyrosine kinases (Abl and Arg) in vascular function. Specifically, loss of endothelial expression of the Abl kinases leads to late-stage embryonic and perinatal lethality in conditional knockout mice, indicating a crucial requirement for Abl/Arg kinases in normal vascular development and function. Endothelial Abl/Arg-null embryos display focal regions of vascular loss and tissue damage, as well as increased endothelial cell apoptosis. An important pro-survival function for the Abl kinases is further supported by our finding that either microRNA-mediated Abl/Arg depletion or pharmacological inhibition of the Abl kinases increases endothelial cell susceptibility to stress-induced apoptosis in vitro. The Abl kinases are activated in response to treatment with the pro-angiogenic growth factors vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). We show that both VEGF- and bFGF-mediated endothelial cell survival is impaired following Abl kinase inhibition.

These studies have uncovered a previously unappreciated role for the Abl kinases in the regulation of the angiopoietin/Tie2 signaling pathway, which functions to support endothelial cell survival and vascular stability. Loss of Abl/Arg expression leads to reduced mRNA and protein levels of the Tie2 receptor, resulting in impaired activation of intracellular signaling pathways by the Tie2 ligand angiopoietin-1 (Angpt1), as well as decreased Angpt1-mediated endothelial cell survival following serum-deprivation stress. Notably, we found that the Abl kinases are activated following Angpt1 stimulation, suggesting a unique dual role for Abl and Arg in Angpt/Tie2 signaling, potentially modulating Tie2 downstream signaling responses, as well as regulating Tie2 receptor expression.

Further, we show an important contribution of the Abl family kinases to the regulation of endothelial permeability responses both in vitro and in vivo. The Abl kinases are activated in response to a diverse group of permeability-inducing factors, including VEGF and the inflammatory mediators thrombin and histamine. We show that inhibition of Abl kinase activity, using either the ATP-competitive inhibitor imatinib or the allosteric inhibitor GNF-2, protects against disruption of endothelial barrier function by the permeability-inducing factors in vitro. VEGF-induced vascular permeability similarly is decreased in conditional knockout mice lacking endothelial Abl expression, as well as following treatment with Abl kinase inhibitors in vivo. Mechanistically, we show that loss of Abl kinase activity is accompanied by activation of the barrier-stabilizing GTPases (guanosine triphosphatases) Rac1 and Rap1, as well as inhibition of agonist-induced Ca2+ mobilization and generation of acto-myosin contractility.

Taken together, these results demonstrate involvement of the Abl family kinases in the regulation of endothelial cell responses to a broad range of pro-angiogenic and permeability-inducing factors, as well as a critical requirement for the endothelial Abl kinases in normal vascular development and function in vivo. These findings have implications for the clinical use of Abl kinase inhibitors.

Normal development and homeostasis require dynamic and tight regulation of epithelial architecture. Abnormal epithelial physiology is associated with various pathological conditions including cancers, and may be induced by changes in epithelial polarity, morphology and/or movement. Among the signaling pathways modulating epithelial physiology are those downstream of integrins and receptor tyrosine kinases (RTKs). Although roles of multiple integrins and RTKs in epithelium homeostasis have been established, the identity of signals regulating the functions of these surface receptors and the pathways connecting them to the regulation of epithelial architecture remain largely unknown. In this dissertation, I have identified the Abl family of non-receptor tyrosine kinases (Abl and Arg) as regulators of beta1 integrin and Met receptor tyrosine kinase signaling.

Abl family kinases are hyper-activated in multiple solid tumors and implicated in epithelial polarity regulation. Dysfunction of beta1 integrin is also associated with carcinoma development. To study the role of the Abl family member Arg in epithelial cell polarity, I have taken advantage of a three-dimensional (3D) cell culture system, where Madin Darby canine kidney type II (MDCKII) cells grown in collagen gels develop into polarized cyst structures. I have found that expression of active Arg kinase results in the formation of cysts with inverted apical polarity and that active Arg modulates epithelial polarity by regulating beta1 integrin and small GTPases pathways. In addition, I have shown that Arg regulates the Rap1-beta1 integrin pathway independently of the Rac1 pathway which promotes basal laminin assembly. I have also found that Abl family kinases function downstream of Met and that Abl kinase hyperactivity correlates with Met activation in a mouse mammary tumor model. Abl kinases are activated by HGF which is the ligand for Met, and active Abl kinases are recruited to the Met receptor and promote its tyrosine phosphorylation. Using fluorescence resonance energy transfer (FRET), I have found that Abl kinases regulate RhoA GTPase activity which contributes to actomyosin contractility induced by Met receptor activation. Further, Abl kinases positively regulate Met-dependent migration and invasion induced by HGF in several breast cancer cell lines.

In conclusion, I have identified novel functions of the Abl kinases in epithelial architecture regulation: modulation of epithelial polarity by targeting beta1 integrin function and promotion of Met signaling required for migration and invasion. This has important implications as it suggests potential roles for Abl kinases in carcinoma initiation mediated by beta1 integrin dysfunction, and development of Abl kinases inhibitors for treatment of cancers driven by hyper-activation of HGF-Met signaling.

Adherens junctions are calcium-dependent cell-cell contacts formed during epithelial morphogenesis that link neighboring cells via cadherin receptors. Coordinated regulation of the actin cytoskeleton by the Rho GTPases is required for the formation and dissolution of adherens junctions, however the pathways that link cadherin signaling to cytoskeletal regulation remain poorly defined. The Abl family of tyrosine kinases have been shown to modulate cytoskeletal reorganization downstream of various extracellular signals including growth factor receptors and integrins.

Here we use pharmacological inhibition and RNA interference to identify the Abl family kinases as critical mediators of cadherin-mediated adhesion. Endogenous Abl family kinases, Abl and Arg, are activated and are required for Rac activation following cadherin engagement, and regulate the formation and maintenance of adherens junctions in mammalian cells. Significantly, we show that Abl-dependent regulation of the Rho-ROCK-myosin signaling pathway is critical for the maintenance of adherens junctions. Inhibition of the Abl kinases in epithelial sheets results in activation of Rho and its downstream target ROCK, leading to enhanced phosphorylation of the myosin regulatory light chain. These signaling events result in enhanced stress fiber formation and increased acto-myosin contractility, thereby disrupting adherens junctions. Conversely, Arg gain-of-function promotes adherens junction formation through a Crk-dependent pathway in cells with weak junctions. These data identify the Abl kinases as a novel regulatory link between the cadherin/catenin adhesion complex and the actin cytoskeleton through regulation of Rac and Rho during adherens junction formation.

Unexpectedly, we identified a requirement for Abl and Crk downstream of Rac in the regulation of adherens junctions. Therefore, Abl functions both upstream and downstream of Rac in regulating adherens junctions, which suggests the possibility of a positive feedback loop consisting of Abl-Crk-Rac.

Finally, we identified the Abl kinases as critical mediators of epithelial cell response to HGF. Pharmacological inhibition of Abl kinase activity resulted in impaired dissolution of adherens junctions downstream of HGF stimulation of the Met receptor. Additionally, we observed decreased phosphorylation of the Met receptor itself, along with Gab1 and Crk, downstream effectors of Met signaling. Taken together, these data suggest a requirement for Abl kinases in both adherens junctions formation and turnover.

Peripheral artery disease (PAD) is nearly as common as coronary artery disease, but few effective treatments exist, and it is associated with significant morbidity and mortality. Although PAD studies have focused on the vascular response to ischemia, studies from our lab indicate that skeletal muscle cells, particularly Pax7-expressing muscle progenitor cells (MPCs), also known as satellite cells, may play a critically important role in determining the phenotypic manifestation of PAD. Here, we demonstrate that genetic ablation of satellite cells in a murine model of PAD resulted in a complete absence of normal muscle regeneration following ischemic injury, despite a lack of morphological or physiological changes in resting muscle. Compared to ischemic muscle of control mice (Pax7WT), the ischemic limb of Pax7-deficient mice (Pax7∆) was unable to generate significant force 7- or 28-days after hind limb ischemia (HLI) in ex vivo force measurement studies. A dramatic increase in adipose infiltration was observed 28 days after HLI in Pax7∆ mice, which replaced functional muscle, a phenotype seen in PAD patients with severe disease. To investigate the mechanism of these adipogenic changes, we first investigated whether a pool of progenitor cells known as fibro-adipogenic progenitors (FAPs) was upregulated and demonstrated an increase in the expression of their canonical marker PDGFRα in Pax7∆ mice. Inhibition of FAPs using the drug batimastat resulted in a decrease in muscle adipose tissue and a corresponding increase in fibrosis. MPCs cultured from mouse muscle tissue failed to form myotubes in vitro following depletion of satellite cells in vivo, and they displayed an increased propensity to differentiate into fat in adipogenic medium. Importantly, this phenotype was recapitulated in patients with critical limb ischemia (CLI), the most severe form of PAD. Skeletal muscle samples from CLI patients demonstrated an increase in adipose deposition in more ischemic regions of muscle, which corresponded with a decrease in the number of satellite cells in those regions. Collectively, these data demonstrate that Pax7+ MPCs are required for normal muscle regeneration after ischemic injury, and they suggest that targeting muscle regeneration may be an important therapeutic approach to prevent muscle degeneration in PAD. Future studies will focus on the role of other supporting cells (such as pericytes) and the cross-talk between FAPs and satellite cells in ischemic muscle regeneration.

Cilia are hair-like structures found on a number of cells serving a variety of different functions. The common denominator between cilia is that they are made up of a microtubule-based projection called the axoneme and nucleated by a modified mother centriole termed the basal body. While there has been an extensive amount of work interrogating ciliogenesis, the requirements for cilia maintenance has less been appreciated. Recent work is also starting to reveal that the cilia biogenesis pathways for these different cilia are different, suggesting that the requirements for their maintenance may also be different.

TTBK2 is a key regulator of primary cilia assembly. In the following study, we identify novel pathways that regulate and are regulated by TTBK2 using BioID. Using tamoxifen-inducible Cre-recombinase cell and mouse systems, we are able to allow these systems to build cilia, deplete TTBK2 levels after cilia formation by tamoxifen induction, assess changes to different types of cilia over time, and validate some of the protein interactions we identified by BioID.

Metastases are common and devastating complications linked to ~90% of cancer deaths. Therapy-resistance is a major challenge for the treatment of cancer cell metastasis as metastatic cells metabolically rewire to survive cytotoxic therapies and adapt to new environments. Understanding and effectively targeting these metabolic changes opens an entirely new therapeutic avenue for combating cancer by defining cancer-related metabolic vulnerabilities. Using a CRISPR/Cas9 loss-of-function screen and RNA-sequencing analysis, the studies presented herein identify two metabolic vulnerabilities that arise following ABL allosteric inhibitor treatment to target metastatic and therapy-resistant cancer cells. First, we identify a novel combination therapy of ABL kinase allosteric inhibitors with lipophilic statins that impairs growth of clinically relevant therapy-resistant and brain metastatic lung cancer cells in vitro and in in vivo using mouse models. We found that ABL allosteric inhibitors impair mitochondria function without altering glycolytic capacity, leading to sensitization to statin therapeutics, and enhanced synergy to promote cancer cell death by combination therapy. Further, we found that ABL inhibitors are sensitized to statins due to the ability of statin therapeutics to inhibit the isoprenoid pathway, specifically protein geranylgeranylation. These results reveal a potential striking clinical benefit as synergy was not noted upon combination with standard of care therapeutics, gefitinib and docetaxel, and identify a new treatment strategy for patients refractory to first-line therapeutics or with metastases to difficult to treat organs like the brain. We have also characterized a novel ABL signaling axis as ABL inhibition was shown to deplete SLC7A11 protein levels in cancer cells. SLC7A11 is the catalytic subunit of system xCT and enables cystine import for cell detoxification and concomitant glutamate export. By depleting cancer cells of SLC7A11, cell detoxification processes are limited and excretion of toxic glutamate levels into the tumor microenvironment decrease. These data suggest that ABL regulation of this pathway could extend survival and relieve harmful symptoms in patients experiencing primary and secondary metastatic tumors. Collectively, our findings reveal metabolic vulnerabilities that can be targeted in cancer cells through treatment with ABL allosteric inhibitors, leading to improved patient survival and quality of life.

Human epidermal growth factor receptor 2-positive (HER2+) and triple-negative breast cancer patients often present with brain metastasis. HER2-targeted therapies have not been successful to treat brain metastases in part due to poor blood-brain barrier (BBB) penetrance and emergence of resistance. Notably, there is a lack of effective FDA-approved targeted therapies for patients with triple-negative breast cancer brain metastases. Here we explore the transcriptional and translational networks that drive breast cancer brain metastasis.

We report that ABL kinase allosteric inhibitors improve overall survival and impair HER2+ brain metastatic burden in vivo. ABL kinase inhibition leads to a profound decrease in HER2 protein levels in HER2+ brain metastatic cells. Mechanistically, ABL kinases regulate translation of HER2/ERBB2 through the RNA binding protein Y-box-binding protein 1 (YB-1). YB-1 crosslinking immunoprecipitation (CLIP)-sequencing and RNA immunoprecipitation of YB-1 revealed YB-1 binds to ERBB2 and a subset of mRNAs linked to brain metastasis. Loss of YB-1 inhibits brain metastatic outgrowth and impairs expression of a subset of ABL-dependent brain metastatic targets. These data support a previously unknown role for ABL kinases in the translational regulation of brain metastatic targets through YB-1 and offer a new therapeutic target for HER2+ brain metastasis patients. To characterize the transcriptional landscape of human brain metastases, spatial transcriptomics was performed on patient samples. Analysis of the tumor and surrounding brain microenvironment may identify novel vulnerabilities that could translate into therapies for patients with brain metastases.

Stem cells are defined by their ability to make more stem cells, a property known as self-renewal and their ability to generate cells that enter differentiation. One mechanism by which fate decisions can be effectively controlled in stem cells is through asymmetric division and the correct partitioning and inheritance of cell fate determinants. While hematopoietic stem cells have the capacity to divide through asymmetric division, the molecular machinery that regulates this process is unknown and whether its activity is required in vivo remains unclear. Here we show that Lis1, a dynein-binding protein and regulator of asymmetric division, is critically required for blood development and for hematopoietic stem cell renewal in fetal and adult life. In particular, conditional deletion of Lis1 led to a severe bloodless phenotype and embryonic lethality in vivo. In both fetal and adult mice, loss of Lis1 led to a failure of normal self-renewal, which included impaired colony-forming ability in vitro and defects in long-term reconstitution ability following transplantation. As a possible mechanism, we find that the absence of Lis1 in hematopoietic cells, in part, accelerates differentiation linked to the incorrect inheritance of cell fate determinants. Furthermore, using a live cell imaging strategy, we find that the incorrect inheritance of cell fate determinants observed following the loss of Lis1 is due defects in spindle positioning and orientation. Finally, using two animal models of undifferentiated myeloid leukemia, we show that Lis1 is critical for the aberrant cell growth that occurs in cancer. Deletion of Lis1 both at the early and late stages of myeloid leukemia blocked its propagation in vivo and led to a marked improvement in survival. Together, these data identify Lis1 and the directed control of asymmetric division as key regulators of normal and malignant hematopoietic development.

The ABL family of non-receptor tyrosine kinases, ABL1 (also known as c-ABL) and ABL2 (also known as Arg), links diverse extracellular stimuli to signaling pathways that control cell growth, survival, adhesion, migration and invasion. ABL tyrosine kinases play an oncogenic role in human leukemias. However, the role of ABL kinases in solid tumors including breast cancer progression and metastasis is just emerging.

To evaluate whether ABL family kinases are involved in breast cancer development and metastasis, we first analyzed genomic data from large-scale screen of breast cancer patients. We found that ABL kinases are up-regulated in invasive breast cancer patients and high expression of ABL kinases correlates with poor prognosis and early metastasis. Using xenograft mouse models combined with genetic and pharmacological approaches, we demonstrated that ABL kinases are required for regulating breast cancer progression and metastasis to the bone. Using next generation sequencing and bioinformatics analysis, we uncovered a critical role for ABL kinases in promoting multiple oncogenic pathways including TAZ and STAT5 signaling networks and the epithelial to mesenchymal transition (EMT). These findings revealed a role for ABL kinases in regulating breast cancer tumorigenesis and bone metastasis and provide a rationale for targeting breast tumors with ABL-specific inhibitors.

The Abl family of protein tyrosine kinases regulates diverse cellular processes by coordinating cytoskeletal rearrangements. Recent data indicate that pharmacological inhibition of Abl kinases reduces inflammation in preclinical models and in the clinic. While a previous role for Abl kinases in lymphocytes had been described, it remained unclear if Abl kinases regulate innate immune function. To explore this possibility, we generated a myeloid-specific conditional Abl knockout mouse. Using a combination of molecular, genetic, and pharmacological approaches, we demonstrate a role for Abl kinases in regulating the efficiency of macrophage phagocytosis and inflammatory responses. Bone marrow-derived macrophages from mice lacking Abl and Arg kinases exhibit inefficient phagocytosis of sheep erythrocytes and zymosan particles. Treatment with the Abl kinase inhibitors imatinib and GNF-2 or overexpression of kinase-inactive forms of the Abl family kinases also impairs particle internalization in murine macrophages, indicating Abl kinase activity is required for efficient phagocytosis. Further, Abl kinases are present at the phagocytic cup and are activated by Fcgamma receptor engagement. The regulation of phagocytosis by Abl family kinases is mediated in part by the Syk kinase. Loss of Abl and Arg expression or treatment with Abl inhibitors reduced Syk phosphorylation in response to Fcgamma receptor ligation. The link between Abl family kinases and Syk may be direct as purified Arg kinase phosphorylates Syk in vitro. Further, overexpression of membrane-targeted Syk in cells treated with Abl kinase inhibitors partially rescues the impairment in phagocytosis.

Our studies also revealed a role for Abl kinases in macrophage and cancer cell invasion. Inhibition of Abl kinases suppressed cell invasion in vitro, whereas overexpression of Abl kinases enhanced extracellular matrix degradation. We found that partial loss of Abl kinase expression in myeloid cells reduced macrophage infiltration into tumors in a mouse model of breast cancer. Furthermore, pharmacological inhibition of Abl kinases reduced myeloid cell infiltration and slowed tumor growth in subcutaneous tumor models. We also found that Abl expression and activity are elevated in subsets of human tumor samples. Taken together, our results suggest Abl kinases have an important role in cancer and inflammation, and represent important therapeutic targets for their treatment.

Angiogenesis, the production of new vessels from pre-existing vasculature, is a complex biological process that is dependent on a series of regulated events, including endothelial cell (EC) proliferation, migration, survival, and capillary morphogenesis (tube formation). These events are required for angiogenesis to occur properly and the steps are regulated by a variety of vascular growth factors and their receptors. Tie2, an endothelial receptor tyrosine kinase (RTK), is required for embryonic and postnatal angiogenesis. Studies have demonstrated that Tie2 is proteolytically cleaved, producing a 75 kDa soluble receptor fragment (sTie2). However, the mechanisms and function of sTie2 have not been elucidated. Here, we investigated signaling pathways and effector molecule(s) responsible for Tie2 cleavage. Additionally, we investigated the role of other growth factors and conditions on the degree of Tie2 cleavage. Finally, we examined sTie2 levels in peripheral artery disease, a human model of ischemic disease. We demonstrated that Tie2 cleavage is VEGF- and PI3K/Akt-dependent and sTie2 can bind Ang1 and Ang2 and prevent ligand-mediated Tie2 activation and downstream cellular responses. Also, ADAM15 cleaves Tie2 in a hypoxia-dependent manner and this response was also observed to be VEGF-mediated. With respect to peripheral artery disease, sTie2 levels were only significantly elevated in the most angiogenically compromised group (critical limb ischemia) of patients. These data shed light on the mechanism and function of Tie2 cleavage and suggest a role for sTie2 in mediating the angiogenic process.

The ABL family of tyrosine kinases are multifaceted signaling molecules that link diverse extracellular stimuli to intracellular signaling pathways that control cell growth, survival, migration, and invasion during development and normal cellular homeostasis. In recent years, it has been established that multiple solid tumor types hijack ABL kinase signaling to support tumor progression and metastasis. The ABL kinases often potentiate these processes by inducing the stability and/or transactivation of transcriptional regulators. Using in vivo mouse models of solid tumor metastasis combined with mechanistic cell signaling and biochemistry, the studies presented herein uncovered multiple transcriptional networks modulated by the ABL kinases through the regulation of their key transcriptional regulators. First, we report on the discovery of an actionable signaling pathway utilized by brain metastatic tumor cells whereby the transcriptional regulator Heat Shock Factor 1 (HSF1) drives a transcriptional program, divergent from its canonical role as the master regulator of the heat shock response, leading to enhanced expression of a subset of E2F transcription factor family gene targets. We find that HSF1 is required for survival and outgrowth by metastatic lung cancer cells in the brain parenchyma. Further, we identify the ABL2 tyrosine kinase as an upstream regulator of HSF1 protein expression and show that the SRC-Homology 3 (SH3) domain of ABL2 directly interacts with HSF1 protein at a noncanonical, proline- independent SH3 interaction motif. Importantly, knockdown as well as pharmacologic inhibition of ABL2 using allosteric inhibitors impairs expression of HSF1 protein and HSF1-E2F transcriptional gene targets. We have recently identified that expression of the master transcriptional regulator of the cellular response of hypoxia, HIF-1α, as well as expression of the oncogenic transcription factor MYC are dependent on ABL kinase activity. The hypoxia inducible factor 1-α (HIF-1α) enables cells to adapt and respond to hypoxia, and the activity of this transcription factor is regulated by several oncogenic signals and cellular stressors. While the pathways controlling normoxic degradation of HIF-1α are well understood, the mechanisms supporting the sustained stabilization and activity of HIF-1α under hypoxia are less clear. We report that ABL kinase activity protects HIF-1α from proteasomal degradation during hypoxia. Using a FACS-based CRISPR/Cas9 screen we identified HIF-1α as a substrate of the Cleavage and Polyadenylation Specificity Factor-1 (CPSF1), an E3-ligase which targets HIF-1α for degradation whose activity is regulated by ABL kinases. Interestingly, MYC was also shown to be a substrate of CPSF1, and its expression was decreased upon ABL inhibition. These studies highlight the importance of the ABL kinases in the activation of oncogenic transcriptional networks driven by HSF1, HIF-1α, and MYC.

Brain metastases are a devastating consequence of lung cancer resulting in significantly increased mortality. Currently, no effective therapies exist to treat brain metastases due to a lack of actionable targets and failure of systemic therapies to penetrate the blood-brain barrier (BBB). Using in vivo mouse models of brain metastasis combined with mechanistic cell signaling and transcriptomic approaches, the studies presented herein identify two actionable signaling pathways required during the colonization of metastatic lung cancer cells in the brain. First, we identify an autocrine AXL-ABL2-TAZ signaling axis in lung adenocarcinoma brain metastasis whereby nuclear accumulation of the TAZ transcriptional co-activator drives expression of ABL2 and AXL encoding protein tyrosine kinases which engage in bidirectional signaling. Activation of the ABL2 kinase in turn results in increased tyrosine phosphorylation of TAZ and enhances TAZ nuclear localization, thereby establishing an autocrine feed forward signaling loop. In addition to driving expression of ABL2 and AXL, TAZ also drives transcriptional activation of a number of neuronal-related gene targets, including L1CAM which was previously shown to promote metastatic colonization and outgrowth in the brain. Importantly, pharmacologic inhibition of the ABL kinases with BBB-penetrant ABL allosteric inhibitors disrupts AXL-ABL2-TAZ signaling and extends survival in brain metastasis-bearing mice. We also characterize an additional transcriptional program driven by Heat Shock Factor 1 (HSF1) and E2F family transcription factors during brain metastasis colonization that is required for survival of lung cancer cells at this organ site. Interestingly, the transcriptional program driven by HSF1 in the setting of brain metastasis is strikingly divergent from the canonical heat shock response, and loss of HSF1 markedly impairs colonization of tumor cells in in vivo models of brain metastasis. We identify ABL2 as a regulator of HSF1 and E2F protein expression and find that inhibition of ABL2 robustly impairs HSF1-E2F target gene expression. Collectively, our findings reveal ABL2 as a central hub of both AXL-ABL2-TAZ signaling and HSF1-E2F-coregulated transcription and provide evidence supporting the use of ABL kinase allosteric inhibitors for the treatment of lung cancer brain metastases.

Epithelial cells are the organism’s first defensive barrier to pathogens, toxins, and other noxious elements. Constant exposure to these elements requires exquisite control over the process of regeneration to ensure that damaged cells are constantly replenished while preventing overgrowth and maintaining organ size. Many of the mechanisms of proliferation and differentiation are exploited by sub-populations of cells following injury but also by tumor cells to allow uncontrolled growth and acquired resistance.

In the lung, elegant studies have identified several putative stem cell populations contributing to the regeneration of lung epithelial cells following injury. However, despite the abundance of region specific lung stem/progenitor cells, a specific cellular source that can be activated in response to damage remains unknown. The studies presented here identify the Abl kinases as a potential cellular target in several progenitor cell types that can be targeted for efficient recovery following a variety lung injuries, including bacterial and viral pneumonia. Furthermore, by targeting some of the same pathways of de-differentiation cancer cells use to acquire resistance, Abl kinase inhibition promotes differentiation of primary lung cancer cells and renders them more susceptible to chemotherapy.

Breast cancers (BC) remain the most lethal malignancies amongst women. Subtype heterogeneity and aggressive invasion are believed to be major contributors for poor outcomes. Triple negative breast cancers (TNBC) are notoriously aggressive, difficult to treat, pro-inflammatory and highly metastatic. Patients that are diagnosed with localized, surgically resectable tumors are treated with neoadjuvant (preoperative) and adjuvant (post-operative) chemotherapy in the hopes of eliminating micro- and oligo-metastatic disease. However, a majority of these patients progress to metastatic disease even under the selective pressure of our current aggressive therapeutic armament. Tumor metastases to tropic organ sites – primarily the lungs, bones, and brain – can wreak havoc on patients with triple-negative breast cancer. Unfortunately, most patients with metastatic TNBC decline quickly. In order to change the tide against pancreatic cancer and other aggressively metastatic tumor types, there need to be new clinical approaches in the arena of anti-metastatic therapeutics.Tumor metastasis is an incredibly complex process involving interactions between tumor cells, the tumor microenvironment (TME), the surrounding stromal tissues, blood and lymphatic vessels, and the innate and adaptive immune systems. The earliest steps of the metastatic cascade require the tumor cells to undergo the epithelial-to-mesenchymal transition (EMT) in order to have the appropriate morphology, detach from the primary tumor site, invade/intravasate into the stroma and reach the host’s blood or lymphatic vessels in order to travel to distant sites. Even once in the circulation, the tumor cell has to survive the turbulent hemodynamic environment before embedding itself into the distant organ site. Various immune sensors also contribute to this complex interplay, especially the toll-like family of receptors (TLRs). These receptors evolved as a part of the innate immune system to detect pathogen-associated molecular patterns (PAMPs) to combat infectious pathogens including bacteria, fungi and viruses. They can also respond to molecular motifs from self – also known as damage-associated molecular patterns (DAMPs). These receptors are typically expressed by immune cells such as macrophages, dendritic cells, and neutrophils but in recent times have been shown to be expressed on both cancer cells and stromal cells within the tumor. The expression and activation of these receptors by either PAMPs and/or DAMPs has been implicated in tumor progression and metastasis in a variety of solid and hematologic malignancies. Going beyond their utility to act as universal antidotes for aptamers (which was already beyond their initial purpose as gene delivery tools), our lab previously discovered that a subset of nucleic-acid binding polymers (NABPs) could behave as nucleic -acid scavengers (NAS). Previously we determined that NASs can block metastatic signals elicited by nucleic acid-containing damage-associated molecular patterns (NA DAMPs) in the pancreatic cancer setting. By behaving as anti-inflammatory compounds scavenging extracellular nucleic acids and associated protein complexes that promote pathological activation of TLRs, our lab demonstrated their efficacy in various murine models of disease including systemic lupus erythematosus, sepsis, and influenza infection. Nucleic-acid mediated TLR signaling also facilitates tumor progression and metastasis in several malignancies, including pancreatic cancer and breast cancer. In the first part of this dissertation, we observe that TNBC cells express TLR9, are responsive to TLR9 ligands, and treatment of TNBC cells with chemotherapy increases the release of such NA DAMPs in culture. Chemotherapy derived and BC-patient derived DAMPs increase TLR9 activation and TNBC cell invasion in vitro; however, treatment with the NAS PAMAM-G3 significantly counteracts such effects. NAS treatment in a spontaneous BC murine model (MMTV-PyMT) also led to diminished lung metastatic burden. Thus, NAS may prove useful for inhibiting pathological processes (e.g. metastasis) and represent a novel combination therapeutic approach. The second part of this dissertation, we focused on a clinical descriptive project whereby we took plasma from breast cancer patients pre- and post-neoadjuvant chemotherapy. Our goal was to get an overview of the immune landscape in these patients and if there was any functional consequence of chemotherapy treatment on invasive potential in vitro. While the work shows promise, it really is laying down the groundwork for better understanding what immune mediators are upregulated upon standard-of-care therapies. The last part of this dissertation covers ongoing projects that I started but was unable to bring all the way to publication. Among these are the optimization of cytokine arrays using monocytes and various sources of in vitro or ex vivo NA DAMPs. I also detail a little of the work we carried out building the immune panels to better track and interrogate antigen-tagged pancreatic cancer cells and where they metastasize as well as the effect of PAMAM-G3 in our murine models of cancer. And finally, a small vignette concerning the role of cancer-associated tumor thrombosis and how we can utilize the highly pro-thrombotic KPC pancreatic cancer mouse model to be imaged via a fluorescence labeled thrombin aptamer.