Chronic Lymphocytic Leukaemia (CLL) cells may depend on B-cell receptor (BCR) signalling as well as other microenvironmental survival signals. A peculiarity of CLL is that cells preserve IgD signalling in the presence of reduced IgM signalling, a pattern mimicking anergic B-cells, and consistent with autoantigen exposure. This aim of this thesis was to examine the differing roles of IgM and IgD in CLL. IgM and IgD expression was examined in CLL cells from peripheral blood (PB) and lymph node (LN). Co-expression of IgM and IgD was common, but levels of expression of IgD and IgM vary independently within these compartments, implying they may have differing roles. Most PB CLL samples underwent calcium (Ca) flux after IgD ligation, with evidence of relative IgM anergy. Incubation of cells for 24h in vitro partially restored IgM Ca flux, further supporting an anergic model. A pro-survival role of the BCR was suggested by the finding that BCR ligation was associated with reduced apoptosis in vitro. Mechanistic differences of IgM and IgD signalling were examined using mass-spectrometry based phosphoproteomics. Six CLL samples were compared to five tonsil controls, and >5000 unique phosphopeptides identified. CLL and tonsil phosphoproteomes were compared. BCR induced changes in phosphoproteins and kinases differed between IgM and IgD ligation, and between CLL and tonsillar B-cells. Recognised and novel pathways included BCR, spliceosome and Notch signalling. Evidence in support of anergic signalling in CLL was observed. Anergic IgM signalling is contrasted with IgD as a dynamic process, different in the tissue compartments of CLL. Phosphoproteomics offers a powerful tool for interrogating intracellular signalling, with phosphorylation networks characterizing pathway topology. BCR signalling in healthy B-cells has not previously been studied using this approach and comparisons with CLL highlight known as well as novel pathways that may well represent novel treatment targets.

Oncolytic adenoviral gene therapy has potential as a novel anti-cancer agent for ovarian cancer. Host immune responses are thought to contribute to its therapeutic effects. However further evaluation has been hampered by the lack of an immunocompetent animal model. This is predominantly because human adenovirus is highly species-specific and replicates poorly in murine cells. The second generation human adenovirus (hAd5) type 5 mutant dl922-947 contains a deletion in the E1A CR2 region which allows it to replicate selectively in cells with Rb pathway abnormalities, a finding observed in >90% of human cancers. Previous work has shown that dl922-947 has considerable activity in ovarian cancer and is more potent than E1A wild-type adenoviruses and the E1B-55K mutant dl1520 (Onyx-015, H101). Unfortunately, like its wild-type counterpart, dl922-947 replicates poorly in murine cells and infectious virion progeny are not generated. Mechanisms for the failure of infectious virion formation remain unclear and have been investigated as part of this project. I have found that murine malignant cells can be infected readily with hAd5 vectors. Both early and late viral genes are transcribed and there is evidence of viral genome replication. However, a profound failure of infective virion production is observed together with low levels of late viral protein expression. Ribosome fractionation assays show reduced viral mRNA loading in murine cells, resulting in failure of translation, especially of late transcripts. Aberrant function of the non-structural L4 protein 100K has been identified as a major hurdle to successful viral replication in murine cells. Ectopic expression of L4 100K promotes translation of viral late mRNA and increases expression of late viral proteins and virion production. However, these increases are only partial.

Clonal diversity in cancer has been proposed as a mechanism underlying patient-to-patient variability in therapeutic response, as well as the variability in the likelihood and the time to relapse of acute myeloid leukemia (AML) and other cancers. As a neoplasm develops it often continues to mutate, diversifying into differing clonal populations. Darwinian evolutionary pressures such as inherent fitness imbalances, immune system interactions, and chemotherapy treatments target sensitive clones and drive competition between the clonal populations; selecting for dynamic and resistant cell lines. In this way clonal diversity is conceivable as an impediment to a complete remission with more populations offering more opportunities for therapy resistance. Bulk next generation sequencing (NGS) is currently used to assess clonal composition in leukemia but requires several broad assumptions be made, which can result in incorrect assessments of diversity. Factors such as differences in zygosity of mutations, convergent evolution, or contamination with wild-type/non-cancerous cells can artificially raise or lower reported variable allele frequencies (VAF), leading to errors in clonal assessments. To examine discrepancies between the actual clonal structure and the clonal structures determined through bulk sequencing we developed a novel method of sampling the cell population to identify concurrent mutations. We first created an in silico model which randomly draws cell samples from a simulated tumor multiple times and calculates the VAF for each mutant allele in each sample. By tracking the correlation of mutations between sample replicates, a clonal composition that is not observable from the bulk NGS VAF becomes apparent. We then created in vitro model tumors from AML cell lines, isolated low cell number samples via flow cytometry, and applied a multiplex/nested PCR protocol with pyrosequencing to quantify VAFs in each sample. Again, by calculating the correlation of mutant alleles between replicates, previously unseen with NGS characteristics of the clonal structure becomes evident. Population sampling analysis may potentially offer a solution for clarifying how we can interpret NGS clonal analyses.

DNA topoisomerase I (TopoI) is an essential enzyme involved in the relief of DNA supercoiling during replication. TopoI plays important role in various DNA events, however the recognition that it is the target of anticancer drug camptothecins (CPTs) led to the rapid growth in this field. CPTs inhibit TopoI during S phase and cause double stranded DNA lesions in rapidly dividing cells. This class of drug is used extensively in oncology clinical settings worldwide. However, resistance to this type of therapy has been found in approximately 70% of the patient population. Current evidence supports that degradation of TopoI by the Ubiquitin Proteasomal Pathway (UPP), and consequent compensation by Topoisomerase II expression may be involved in imparting drug resistance, but this mechanism requires much greater understanding. Protein-protein interaction studies have indicated that, BRCA1 is the E3 ligase for TopoI ubiquitination in response to CPT. BRCA1 impaired cells fail to ubiquitinate and degrade TopoI and are sensitive to CPTs. It is important to note that triple negative breast cancer patients have impaired BRCA1 function, higher mutation rate and/or a lower expression of BRCA1. The Bharti lab has shown that TopoI associates with BRCA1. Our work attempts to elucidate the nature of the interaction between BRCA1 and TopoI in the hope of better understanding the mechanism of resistance to camptothecin therapy in TNBC.

<p> <b>Objective:</b> To explore patient factors that influence patients&rsquo; decisions about Neoadjuvant chemotherapy (NC) including patient unmet needs, challenges in decision making, and the role of family and caregiver involvement in treatment decision making. Our goal is to elucidate patient factors including their beliefs, values, expectations, worries about NC and MIBC, emotional challenges, and the importance of caregivers&rsquo; involvement in decision making and selection of treatment procedures. </p><p> <b>Design:</b> A qualitative study with semi-structured single interviews and focus groups. Atlas.ti software was used to analyze the qualitative data collected. </p><p> <b>Settings:</b> Icahn School of Medicine at Mount Sinai (ISMMS) and Northwell Health (NH), New York, USA. </p><p> <b>Methods and Materials:</b> 20 MIBC survivors from the ISMMS (n = 10) and NH (n = 10) were enrolled in this study from September 2015 and August 2016. 17 of them finished a semi-structured single interview and 3 of them joined a focus group interview to share their decision-making process about their treatment plans. Three ISMMS health care providers were interviewed to understand physicians&rsquo; standpoints on the challenges, barriers, and potential facilitators for MIBC patients to make a decision about their treatment plans. </p><p> <b>Results:</b> Patients decisions on whether or not to go through NC were influenced by three major parts: physician&rsquo;s opinion or suggestion, family and spouses&rsquo; opinion, and patients&rsquo; own value and preferences. Sufficient information about treatment efficacy (i.e. treatment options, survival rates, experience sharing from former patients) would benefit patients&rsquo; decision-making process. Therefore ensuring information available for patients is important Results also showed that having a second opinion, experience shared by other patients, and caregivers&rsquo; help with information search helped address some of the patients&rsquo; reported informational needs. Physicians&rsquo; recommendations and advice were reported as influential factors in patients&rsquo; treatment decision making. Physicians&rsquo; challenges confirmed by interviewed patients suggested several areas await improvement including more help with daily activities from healthcare professionals, good bedside manners and professional figures, and opportunity to communicate with former patients. </p><p> <b>Conclusions:</b> Meeting patients&rsquo; informational and emotional needs is imperative to facilitate their decision-making process. Caregivers play significant roles in treatment decision making and recovery. Preparing caregivers for treatment decision making and patient care can maximize support received from the patient&rsquo;s social network. </p><p>

Matrix Metallopeptidase 1 (MMP-1) expression has repeatedly been correlated to tumorigenesis and metastasis. Yet, MMP-1 regulation in a metastatic context remains largely unknown. Here we confirm differential MMP-1 expression in mammary carcinoma cells with varied metastatic potentials and identify a mechanism differentially regulating MMP-1. We show that MMP-1 expression is regulated by an AP-1 element in its promoter in highly metastatic MDA-MB-231 mammary carcinoma cell derivatives. Fra-1, an AP-1 family transcription factor, differentially binds this element in highly metastatic derivatives compared to low-metastatic cells and is required for MMP1 expression. Fra-1 mRNA levels are unchanged in the cell variants, however its protein levels are higher in the metastatic cells. There was no change in protein degradation rates, while protein synthesis rates of Fra-1 increased. These results suggest that protein translation of Fra-1 is differentially regulated in these cells. Consistent with the importance of Fra-1 for tumor growth, we found that Fra-1 overexpression is sufficient to increase cell motility and anchorage independent growth. These results suggest that Fra-1 regulation is critical for regulation of MMP-1 and metastasis.

Acute lymphoblastic leukemia (ALL) is an aggressive hematological cancer which arises from the malignant transformation of B-cell or T-cell progenitors. Despite recent pioneering improvements in intensified combination chemotherapy, 20% of pediatric and 50% of adult ALL patients present with primary drug-resistant leukemia or develop relapse. Treatment of refractory and relapsed ALL has remained a significant clinical challenge with survival rates following relapse of only 40%, highlighting the need to understand the mechanisms which drive drug resistance and relapse of ALL. Through extensive sequencing analyses of matched diagnostic, remission and relapsed DNA samples from patients with B-precursor ALL (B-ALL) and T-cell ALL (T-ALL) we have identified recurrent relapse-specific gain-of-function mutations in the cytosolic 5'-nucleotidase II (NT5C2) gene in 25% of relapsed T-ALLs and 6% of relapsed B-ALLs. NT5C2 is a highly conserved, ubiquitously expressed enzyme which regulates intracellular purine nucleotide levels by dephosphorylating purine monophosphates. NT5C2 also dephosphorylates key metabolites in the activation of purine analog prodrugs such as 6-mercaptopurine and 6-thioguanine which are routinely used in the treatment of ALL, allowing purine analog nucleosides to be readily exported out of the cell. Here we show that mutant NT5C2 proteins have increased 5’-nucleotidase activity and confer resistance to 6-mercaptopurine and 6-thioguanine chemotherapy when expressed in leukemic cells. Consistently, NT5C2 mutations correlate with early relapse and relapse while under therapy. We present a novel T-ALL conditional inducible knock-in mouse model of the highly recurrent NT5C2 R367Q mutation and show that expression of one Nt5c2 R367Q allele from the endogenous locus in primary T-ALL lymphoblasts induces overt resistance and disease progression under therapy with 6-mercaptopurine in vivo, while surprisingly conferring reduced growth and decreased leukemia initiating activity in the absence of chemotherapy. Metabolically we show that the observed loss of fitness in Nt5c2 R367Q tumors can be explained by a severe depletion of endogenous purine monophosphate metabolites as a result of increased Nt5c2 5’-nucleotidase activity. Consistently, using ultra-sensitive mutation analyses we show that relapse-associated NT5C2 mutations are not detectable at initial disease presentation, indicating that NT5C2-mutant tumor cells are negatively selected by clonal competition in the early stages of disease development and only positively selected under prolonged 6-mercaptopruine chemotherapy which is the backbone treatment for ALL following remission. Our findings present the first known example of chemotherapy resistance and disease progression driven by a tumor clone with decreased leukemia initiating activity, highlighting the intense selective pressure of chemotherapy in the clonal evolution of tumors from diagnosis to relapse. Through extensive biochemical and structural characterizations of recombinant NT5C2 mutant proteins, we have grouped relapse-specific NT5C2 activating mutations into 3 different classes, each conferring unique enzymatic behavior in basal conditions and in response to allosteric activation, and each with unique structural features which mediate increased 5’-nucleotidase activity. Moreover, we identify a novel auto-regulatory switch-off mechanism of the NT5C2 enzyme involving movement of an unstructured flexible loop, and present the first crystal structure view of the NT5C2 C-terminal acidic tail, implicating it as an auto-inhibitory brake to the allosteric activation of the enzyme. The presence of multiple mutational mechanisms of activating such a highly conserved enzyme, especially in light of the inherent loss of fitness to the tumor cells, indicates a strong convergent evolution towards activating NT5C2. This is supported by our discovery that patients can harbor multiple leukemic clones with NT5C2 mutations at relapse. Overall our findings highlight NT5C2 as a major driver of drug resistance and relapse of ALL and pinpoint metabolic susceptibilities which could be exploited therapeutically to target NT5C2-mutant tumors in the future. Our in-depth structural and enzymatic knowledge of mutant NT5C2 proteins will serve as an essential tool in the rational drug development of novel NT5C2 inhibitors with increased specificity and selectivity for mutant NT5C2, while our novel Nt5c2 R367Q knock-in mouse model will serve as a platform for the pre-clinical testing of both NT5C2 inhibitors and alternative compounds selective for Nt5c2-mutant leukemias which can be used for prevention and treatment of relapsed ALL.

Ineffective pain management can prolong a patient's length of stay and increase cost of care. Inadequate pain control decreases the patient's quality of life and contributes to poor health outcomes. A recent record audit showed that documentation of pain reassessments occurred only 20% of the time within an hour after administering pain medication. Furthermore, nurses may have insufficient knowledge regarding pain assessments and reassessments or hold irrational fears about addiction leading to inadequate treatment of pain. The purpose of this quality improvement project was to evaluate the effectiveness of an educational program for acute care nurses for the assessment and management of the adult hospitalized patient experiencing pain. Guided by Knowles theory of adult learning, nurses' knowledge regarding pain, assessments, and pain reassessments were evaluated before and after the pain management education program using the Knowledge and Attitude toward Pain Survey (KAPS). A convenience sample of 34 nurses completed the KAPS before and after an educational program addressing pain assessments. Results of the t-test analysis revealed a statistically significant (t = -15.8, df = 33, p<0.00) increase in KAPS scores, from an average pretest score of 70% to an average posttest score of 94%. The results of this project are consistent with the literature, and they illustrate the importance of improving nursing practice by providing nurses with education regarding pain assessments and reassessments as a strategy to improve the management of patients' pain and, resultantly, increase patients' quality of life.

L’érythropoïèse désigne l’ensemble des phénomènes de prolifération et de maturation qui permettent d’obtenir les globules rouges à partir de cellules souches médullaires. C’est un processus de différenciation continue qui génère des cellules érythroïdes de plus en plus matures. L’érythropoïétine (EPO) est le principal régulateur de l’érythropoïèse. En effet, il est indispensable à la survie et à la prolifération des progéniteurs érythroïdes et des précurseurs érythroblastiques. Le rôle des Pims kinases dans la régulation de l'érythropoïèse n’a pas été étudié bien qu’il ait été montré que les souris invalidées pour les trois gènes de la famille Pim présentent une anémie. De plus, une forte diminution du nombre de progéniteurs érythroïdes est observée chez les souries invalidées pour le gène Pim2 et Pim1, suggérant l’importance de cette kinase dans l’érythropoïèse murine. Aucune étude n’a été réalisée pour comprendre le rôle des PIMs dans l’érythropoïèse humaine. Nous avons donc cherché à comprendre l'implication des PIMs kinases dans l'érythropoïèse normale humaine. Nous avons pu montrer que les PIMs kinases sont exprimées dans des érythroblastes humains normaux. En effet, elles sont fortement exprimées en début de différenciation et leur expression diminue au cours de la maturation terminale. Nous avons mis en évidence que l'expression des trois PIMs est régulée au niveau transcriptionnel par l'EPO. Nous avons également montré que les PIMs kinases régulent positivement la prolifération des cellules érythroïdes immatures. Nous avons aussi observé qu'elles soutiennent l'expression du récepteur de la transferrine (TFR1) à la surface des érythroblastes immatures. TFR1 est important pour les érythroblastes car il permet de faire entrer le fer dans ces cellules, où il est intégré dans l’hème permettant la synthèse de l'hémoglobine. L'hémoglobine est nécessaire à la fonction du globule rouge dans le transport de l'oxygène ainsi que pour l'oxygénation des cellules. Ensuite, nous avons voulu étudier le rôle de chacune de ces trois kinases dans les érythroblastes. Nous avons mis en évidence que la kinase PIM2 joue un rôle clef dans l'érythropoïèse normale en étant indispensable à la survie des cellules érythroïdes. Au niveau moléculaire, l’inhibition de PIM2 ne modifie pas la balance des facteurs pro et anti-apoptotiques mais entraîne des dommages à l’ADN. L'apparition de ces dommages est suivie d'une induction de l'apoptose cellulaire. Dans une pathologie comme la Maladie de Vaquez (MV), nous avons mis en évidence que l'inhibition des PIMs diminue le nombre et la taille des colonies érythroïdes, entraîne la diminution de prolifération cellulaire et aussi, pour certains patients, l'apoptose de leurs cellules. Nos résultats mettent donc en évidence que les PIMs sont importantes dans l’érythropoïèse normale humaine et que les inhibiteurs des PIMs pourraient être un traitement alternatif aux traitements classiques dans la MV. / Erythropoiesis encompasses both the proliferation and maturation of bone marrow stem cells, leading to the production of Red Blood Cells (RBCs). It is a process of continuous differentiation that generates more and more mature erythroid cells. Survival and proliferation of erythroid progenitors and erythroblastic precursors is dependent on the cytokine Erythropoietin (EPO), which is the main regulator of erythropoiesis. The PIMs kinases are proto-oncogenes involved in survival and proliferation, whose family is comprised of three members in Humans. The role of PIMs kinases in the regulation of erythropoiesis has not been studied, although it has been shown that mice knocked out for all three genes of the PIM family have anemia. In addition, a sharp decrease in the number of erythroid progenitors is observed in mice knocked out for both the Pim2 and Pim1 gene. All in all, it suggests the importance of these kinases in murine erythropoiesis, and may be also relevant in human erythropoiesis. Therefore, we sought to understand the involvement of PIMs kinases in normal human erythropoiesis. We have been able to show that PIMs kinases are expressed in normal human erythroblasts. Indeed, they are strongly expressed at the beginning of the erythroid differentiation, and their expression decreases during terminal maturation. We have shown that the expression of the three PIMs kinases is regulated at the transcriptional level by EPO. We also have shown that PIMs kinases positively regulate the proliferation of immature erythroid cells. We observed that they support the expression of the transferrin receptor 1 (TFR1) on the surface of immature erythroblasts. TFR1 is crucial for erythroblasts as it allows iron to enter these cells, where it is used for hemoglobin synthesis. Hemoglobin is absolutely required for RBCs function as oxygen transporters, responsible for the oxygenation of the whole organism. Then, we wanted to study the role of each of these three kinases in erythroblasts. We have shown that PIM2 kinase plays a key role in normal erythropoiesis by being essential for the survival of erythroid cells. At the molecular level, inhibition of PIM2 does not alter the balance of pro- and anti-apoptotic factors, but results in damages to DNA. The appearance of these damages is followed by an induction of cellular apoptosis. In a pathology such as Polycythemia Vera (PV), we have demonstrated that the inhibition of PIMs decreases both the number and the size of erythroid colonies. It also slows down cell proliferation, and for some patients, it induces cellular apoptosis. Our results highlight that PIMs are important in human normal erythropoiesis and that inhibitors of PIMs could be an alternative to conventional treatments in PV.

No description available.