Hematopoietic stem cells (HSCs) that function to maintain the hematopoietic compartment through self-renewal and differentiation capacities, as well as their downstream progeny, are susceptible to transformation resulting in the generation of the leukemic stem cell (LSC). Chief in the factors that control HSC regulation and protection of the HSC compartment is the cellular redox environment. Deregulation of the Hematopoietic Stem/Progenitor Cell (HSPC) redox environment results in loss of HSPC function and exhaustion. The characteristic developments of HSPC exhaustion via exposure to redox stress closely mirror phenotypic traits of hematopoietic malignancies, presenting the HSPC/LSC redox environment as a potential therapeutic target. While myelosuppression and HSPC exhaustion are detrimental side effects of classical chemotherapies, new approaches that differentially modify the HSPC/LSC redox environment may demonstrate LSC cytotoxicity while offering protection of normal HSPC function via differential activation of internal signaling pathways. Precisely how the redox environment and downstream signaling events are affected by these treatments remains unclear; thus highlighting the need for robust methods that evaluate the HSPC/LSC redox state. Because the glutathione (GSH), glutathione disulfide (GSSG) redox couple heavily contributes to the management of HSPC function and redox environment, characterizing the GSH/GSSG redox potential at the HSPC level would provide great insight for therapeutic opportunities. However, accurate measurement the GSH/GSSG redox potential within HSPCs/LSCs has been difficult due to their inherently low numbers. Here, we describe the development and validation of a sensitive method used for the direct and simultaneous quantitation of both oxidized and reduced GSH via LC-MS/MS. We use these methodologies to establish a difference in GSH-GSSG half-cell reduction potentials between normal and malignant HSPCs and examine the therapeutic effect of a redox active MnSOD mimetic, Mn(III) mesotetrakis (N-n-butoxyethylpyridinium-2yl) porphyrin, MnTnBuOE-2-PyP5+ (MnP), within these populations in vitro as well as within a human xenograft model in vivo. MnP demonstrates significant cytotoxic effects in several malignant models, while inducing an opposite cytoprotective effect in normal HSPC populations. The GSH/GSSG redox balance, specifically managed by glutathione reductase activity, is identified as a determining factor of MnP efficacy in various malignant populations. Treatment of the human myelodysplastic cell line (MDSL) offers mechanistic insights into MnP efficacy through hydrogen peroxide mediated activation of activator protein 1 (AP-1) signaling. We identify the redox dependent activation of JunB, a known regulator of normal myeloid lineage HSC proliferation, as a transcriptional mechanistic mediator of MnP treatment induced AP-1 signaling resulting in malignant cytotoxicity. The development of this novel method allowing for the identification of targetable differences between normal and malignant cell populations has provided insight to the underpinnings of potential redox based therapies. Additionally, the finding that MnP can target varying cellular redox states and exert selective cytotoxicity in malignant over normal populations by re-gaining lost control of AP-1 signaling demonstrates the potential for development of safe therapeutics within a variety of clinical applications.

Acute myeloid leukemia (AML) is the second most common type of leukemia and accounts for 80% of adult acute leukemia cases and is characterized by the accumulation of poorly or undifferentiated myeloid blast cells. Standard treatment includes chemotherapy, which if unsuccessful, is followed by more rigorous chemotherapy as well as stem cell transplantation. Considering most patients are over the age of 45, these more rigorous therapies are not always possible, and as such, new therapies must be developed. Furthermore, AML patients harboring a chromosomal rearrangement involving Multiple Lineage Leukemia (MLL) that results in the expression of an MLL fusion protein exhibit far worse prognoses than patients without. In recent years, Danio rerio (zebrafish) has emerged as a powerful model organism for investigating human blood malignancies due to the conservation of hematopoiesis between humans and zebrafish. The first objective of this study was to develop a transient transgenic AML model in zebrafish, and the second objective was to determine if co-treatment with two medications currently in human trials for AML, Venetoclax and Flavopiridol, would be more effective than using either drug individually. In order to develop a transient transgenic AML model, we first developed a DNA construct encoding a known mixed lineage leukemia (MLL) fusion protein associated with human AML, MLL-ENL, driven by the zebrafish lysozyme C (lyz) promoter, which drives myeloid specific expression in zebrafish. We then microinjected single-cell zebrafish embryos with DNA encoding lyz driven MLL-ENL along with transposase mRNA to facilitate the genomic integration of MLL-ENL. Injected embryos were first tested for MLL-ENL expression, and subsequently tested for AML phenotypic characteristics, via whole mount in-situ hybridization (WISH) at 72 hours post fertilization (hpf). First, WISH analysis utilizing a human MLL riboprobe verified MLL-ENL expression in injected embryos, and WISH analysis utilizing the same MLL riboprobe revealed an expansion and clustering of MLL positive cells in injected embryos, characteristic of an AML phenotype. Embryos injected with MLL-ENL DNA were then treated with either DMSO (vehicle), 200 nanomolar (nM) Venetoclax, 200 nM Flavopiridol, or 200 nM Venetoclax and 200 nM Flavopiridol from 24 hpf to 72 hpf. MLL WISH analysis of injected and treated embryos revealed a reduction in MLL positive cells in both Venetoclax treated embryos and Flavopiridol treated embryos, and an even greater reduction in MLL positive cells in embryos treated with both Venetoclax and Flavopiridol, compared to controls. Although further analysis is required to be confident, these data suggest that we successfully developed an AML transient transgenic model in zebrafish. Furthermore, these data suggest that Venetoclax and Flavopiridol co-treatment could yield better outcomes for AML patients than treatment with either drug individually.

High expression of B-Myb (encoded by MYBL2), an oncogenic transcription factor, is associated with cell cycle deregulation and poor prognosis in several cancers, including ovarian cancer. However, the mechanism by which B-Myb alters the cell cycle is not fully understood. In proliferating cells, B-Myb interacts with the MuvB core complex including LIN9, LIN37, LIN52, RBBP4, and LIN54, forming the MMB (Myb-MuvB) complex, and promotes transcription of genes required for mitosis. Alternatively, the MuvB core interacts with Rb-like protein p130 and E2F4-DP1 to form the DREAM complex that mediates global repression of cell cycle genes in G0/G1, including a subset of MMB target genes. Here, we show that overexpression of B-Myb disrupts the DREAM complex in human cells, and this activity depends on the intact MuvB-binding domain in B-Myb. Furthermore, we found that B-Myb regulates the protein expression levels of the MuvB core subunit LIN52, a key adaptor for assembly of both the DREAM and MMB complexes, by a mechanism that requires the S28 phosphorylation site in LIN52. To validate our cellular findings, we determined the effect of B-Myb levels on DREAM target gene expression in HGSOC tissue samples and corresponding patient outcomes. Given that high expression of B-Myb correlates with global loss of repression of DREAM target genes in breast and ovarian cancer, our findings offer mechanistic insights for aggressiveness of cancers with MYBL2 amplification and establish the rationale for targeting B-Myb to restore cell cycle control.

According to the American Cancer Society, it has been estimated that in 2019 alone, there will be approximately 53,000 new cases of oropharyngeal cancers. Oropharyngeal cancers are the largest subset of head and neck squamous cell carcinomas (HNSCCs), which are the sixth most common cancer across worldwide populations. They, along with other HNSCCs, fall under a category of cancers known as Human papillomavirus (HPV)-associated cancers, and it has been found that upwards of 70% of these cancers can be attributed to high-risk HPV infections. Specifically, the high-risk HPV gene, E7, plays a key role in relieving cell cycle repression by disrupting the DREAM complex via competitive binding with p130, driving the cell cycle and cell proliferation. In order to combat this interaction, a LIN52-S20C mutation was developed, in hopes of reducing E7 binding of p130 and stabilizing the DREAM complex. We utilized human cervical cell lines, immortalized keratinocytes, and mouse fibroblasts, all of which contained the HPV16 genome, as models to observe the effects of the LIN52-S20C mutation on HPV-mediated hijacking of the cell cycle. Not only were we able to replicate the increased proliferation and upregulated DREAM gene expression in infected cells, but we were also able to observe some reversal of these effects in many of our cell models through the expression of the LIN52-S20C variant. The findings of these studies have been promising and provide a basis for future works, and we hope that the effects of the LIN52-S20C mutation can be translated into studies in in vivo models.

Transient ablation of regulatory T cells has been shown to be effective at hindering tumor growth and metastasis in murine breast cancer model. Based on our lab’s previous work, we have demonstrated that NK cells and CD8+ cytotoxic T cells were not required for the protective effect of Treg cell ablation. However, we also reported that CD4+ helper T cells and IFN-γ were required for the protective effect of Treg cell ablation. Furthermore, we observed that CD11B+ cells responded to Treg ablation therapy by up-regulating target genes of IFN-γ. Therefore, this study aimed to investigate the connection between the myeloid cell compartment and IFN-γ signaling after regulatory T cell ablation therapy. Through a combination of conditional knockout mouse models, cellular fate mapping experiments, adoptive transfers, and co-injection experiments, we demonstrated that tumor-associated macrophages (TAMs), derived from the bone marrow via CCR2/CCL2 axis, were responsible for the therapeutic effect of regulatory T cell ablation. In addition, we determined that IFN-γ signaling was required for the TAMs to mediate the protective phenotype seen after regulatory T cell ablation. Furthermore, based on our findings, we developed a genetic signature based on TAMs from treated or untreated tumors that had a predictive value for patient survival. Thus, our findings indicated a strong connection between IFN-γ release, classical activation of TAMs, and depletion of regulatory T cells, and taken together, our data could offer potential clinical strategies to mimic regulatory T cell ablation.

Prolactin (PRL) and its receptor (PRLr) have been implicated in the development and progression of human breast cancer. PRL activates its receptor and induces activation of proximal Janus kinase 2 (Jak2) for signal transduction. Here, we sought to determine the role of PRLr-associated peptidyl-prolyl isomerase, cyclophilin A (CypA), in modulating structure/function relationships of the PRLr. It was demonstrated that CypA mediated PRL-induced conformational change of the CFP- and -YFP tagged forms of the PRLr cytoplasmic-tail, whereas CypA inhibition by NIM811 (N-methyl-4-isoleucine cyclosporin) or knockdown blocked the conformational change of the PRLr assessed by Fluorescence Resonance Energy Transfer (FRET) signal or efficiency. To further investigate the consequences of CypA inhibition or knockdown on the PRLr/Jak2 complex mediated signaling/functions, analyses of phospho-tyrosine residues that are believed to be important for interactions/signaling were investigated. It was found that NIM811 inhibition or CypA shRNA knockdown significantly reduced prolactin-stimulated phosphorylation of PRLr/Jak2 intermediates and their association with the PRLr in breast cancer cells. A microarray analysis revealed that NIM811 inhibited approximately 66% of the top 50 PRL-induced genes. NIM811 inhibited breast cancer cell proliferation, survival, migration and anchorage-independent growth. Subsequent NIM811 treatment of a triple negative breast cancer xenograft inhibited primary tumor growth, outgrowth of macro-metastasis and induced central tumor necrosis. Furthermore, loss of CypA in the MMTV-PyMT mouse model demonstrated inhibition of tumorigenesis with significant reduction in lung and lymph node metastasis. Overall, CypA modulates PRL-induced conformational change of the C-terminus of the PRLr through its isomerase activity, altering PRLr/Jak2 complex signaling/functions in breast/mammary tumorigenesis and metastasis.

No description available.

In the last four years, seven new drugs have been FDA approved for the treatment of late stage melanoma, for the field of melanoma, this marks an incredibly exciting. Three of these new therapies, vemurafenib, dabrafenib and trametinib are small molecule kinase inhibitors that target the MAPK pathway and as such have been approved for the treatment of BRAFV600 mutant melanomas. Yet despite recent advances, mechanisms of intrinsic and acquired BRAF inhibitor resistance continue to undermine uniform and long-lasting therapeutic responses. Several studies have shown that the reactivation of MAPK signaling is a critical event leading to BRAF inhibitor resistance. These studies lead to the evaluation and subsequent FDA approval of frontline BRAF (dabrafenib) plus MEK (trametinib) inhibitors to delay drug resistance. Though this approach has meaningful clinical benefit, there are still a number of patients who do not respond to therapy or who, through unknown mechanisms, succumb to refractory disease. In an effort to identify drivers of MAPK inhibitor resistance, several studies have relied on traditional genomics methods. While gene-based approaches have guided precision medicine, they do not address the dynamics of the global signaling changes that occur following acquired resistance. The dissertation herein will describe our efforts to fill these gaps of knowledge and will expand upon the evolution and development of our understanding of intrinsic and acquired MAPK pathway inhibitor resistance. This work will elaborate on our early understanding of single agent BRAF inhibitor resistance, the use of genomic and proteomic approaches to further elucidate these mechanisms, and evidence based approaches to delay and overcome single agent BRAF inhibitor resistance. This work will describe global phosphoproteomic and bioinformatics methodologies to elucidate the underlying processes of both single (BRAF) and dual agent (BRAF plus MEK) inhibitor resistance as well as strategies to constrain dual agent BRAF plus MEK inhibitor resistance.

This dissertation is devoted to the study of the molecular biology of major tumor suppressors, defined as those that prevent the cellular processes identified as the hallmarks of cancer. Specifically, the major tumor suppressors pRb and STK11 are explored in the context of osteosarcoma and lung cancer, respectively. RB1 was the first tumor suppressor gene discovered. Over four decades of work have revealed that the Rb protein (pRb) is a master regulator of biological pathways influencing virtually every aspect of intrinsic cell fate including cell growth, cell-cycle checkpoints, differentiation, senescence, self-renewal, replication, genomic stability and apoptosis. While these many processes may account for a significant portion of RB1's potency as a tumor suppressor, a small, but growing stream of evidence suggests that RB1 also significantly influences how a cell interacts with its environment, including cell-to-cell and cell-to-extracellular matrix interactions. Chapter 2 highlights pRb's role in the control of cell adhesion and how alterations in the adhesive properties of tumor cells may drive the deadly process of metastasis. Chapter 3 defines a role for pRb as a suppressor of the progression to metastasis by upregulating integrin α10. Transcription of this integrin subunit is herein found to be pRb-dependent in mouse osteoblasts. Classic pRb partners in cell cycle control, E2F1 and E2F3, do not repress transcription of integrin α10 and phosphorylation of pRb is not necessary for activation of the integrin α10 promoter. Promoter deletion revealed a pRb responsive region between -108bp to -55bp upstream of the start of the site of transcription. pRb activation of transcription also leads to increased levels of integrin α10 protein and a greater concentration of the integrin α10 protein at the cell membrane of mouse osteoblasts. These higher levels of integrin α10 correspond to increased binding to collagen substrate. Consistent with our findings in mouse osteoblasts, we found that integrin α10 is significantly underexpressed in multiple solid tumors that have frequent inactivation of the pRb pathway. Bioinformatically, we identified data consistent with an 'integrin switch' that occurs in multiple solid tumors consisting of underexpression of integrins α7, α8, and α10 with concurrent overexpression of integrin β4. pRb promotes cell adhesion by inducing expression of integrins necessary for cell adhesion to a substrate. We propose that pRb loss in solid tumors exacerbates aggressiveness by debilitating cellular adhesion, which in turn facilitates tumor cell detachment and metastasis. Lung cancer is the leading cause of cancer-related death in the U.S. and additional targeted therapies are desperately needed to treat these patients. STK11 is the third most frequently mutated gene in lung adenocarcinoma following only KRAS and TP53, yet its mutational status is not currently clinically evaluated and no therapies have been approved to specifically target its pathway. A deep understanding of the complex pathways controlled by STK11 and their alterations in cancer are required to develop effective therapies for patients with loss-of-function mutations. In Chapter 4 we present the current understanding of STK11, focusing on its molecular biology and therapeutic implications, including a compilation of studies evaluating STK11 somatic mutations in human lung cancer tissue and how the frequency of these mutations varies across histological subtypes and patient populations. Finally, we review the strategies being used to target STK11-deficient cancers at the clinical trial, pre-clinical, and basic science levels as well as proposing potential new therapies that might benefit this patient population. STK11 is a tumor-suppressor commonly mutated in lung adenocarcinoma (LuAd). There are a number of agents that may selectively target the deregulated pathways in STK11 mutated tumors, and thus, identifying the subset of adenocarcinomas that harbor these mutations could have significant clinical benefit. In Chapter 5, we characterized a cohort of 442 adenocarcinoma patients with respect to STK11 mutation status and subset of this cohort using immunochemistry, gene expression, and western blotting. We found that measuring STK11 mutation status is complicated by the fact that many STK11 mutations lead to expression of a stable protein that is indistinguishable from wild type (WT) via immunohistochemistry. To circumvent this, we used published cell line mutation and gene expression data to derive a signature correlating with STK11 mutation status. This signature was validated in the cohort of 442 lung adenocarcinomas and strongly correlates with mutation status (ROC curve AUC = 85.29). These data suggest that STK11; mutation status may be best assessed by measuring the downstream targets included in our signature.

Histone modifying enzymes and chromatin remodeling complexes play an important regulatory role in chromatin dynamics that dictate the interaction of regulatory factors involved in processes such as DNA replication, recombination, repair and transcription, with DNA template. The CHD (Chromodomain Helicase DNA Binding Protein) family of proteins is known to be involved in the regulation of gene expression, recombination and chromatin remodeling via their chromatin specific interactions and activities. Phenotypic analysis of the Chd2 mutant mouse model developed by our laboratory indicates that the Chd2 protein plays a critical role in tumor suppression as the heterozygous mutant mice develop spontaneous lymphomas. In this study we demonstrate that mutation of Chd2 renders cells susceptible to inefficient DNA repair and genomic instability. Homozygous and heterozygous Chd2 mutant mouse embryonic fibroblast accumulates higher levels of gamma-H2AX after DNA damage. Chd2 mutant cells show inefficiency in DNA repair of DNA lesions induced by X-rays and UV irradiation as assessed by single cell gel electrophoresis assays. These cells also exhibit increased chromosomal aberrations after treatment with low doses of X-ray irradiation (2 Gy) and show increased radiosensitivity in a clonogenic survival assay. At the molecular level, endogenous CHD2 protein level is induced after exposure to X-ray radiation. In addition, we have also demonstrated in this study that CHD2 is phosphorylated after DNA damage and is a potential substrate for phosphoinositide 3-kinase-related kinases (PIKK) - ATM/ATR. Additionally, mass spectrometric analysis showed possible association of CHD2 with the paraspeckle family of proteins known to be involved in an array of cellular processes specifically in RNA processing and DNA repair. An in vivo splicing assay demonstrated that CHD2 played a role in modulation of pre-mRNA splicing event. Collectively, our findings suggest that CHD2 is a multi-functional protein working with the paraspeckle protein complex to facilitate both the pre-mRNA splicing process and the initial DNA repair process. CHD2 may also be involved in the later stages of DNA damage response pathway by influencing p53’s transcriptional activity.