Medulloblastoma (MB) is the most common malignant pediatric brain tumor. Out of the four molecular subgroups (WNT, SHH, Group 3 and Group 4), Group 3 patients face the highest incidence of leptomeningeal spread and overall patient survival of less than 50%. Current clinical trials for recurrent MB patients based on genomic profiles of primary, treatment-naïve tumors, provide limited clinical benefit since recurrent metastatic MBs are highly genetically divergent from their primary tumors. The paucity of patient matched primary and recurrent MB samples has contributed to the lack of molecular targets specific to medulloblastoma recurrence, limiting relapsing MB patients to palliation. Our previous in silico analyses revealed enriched expression of many stem cell self-renewal regulatory genes in Group 3 MB. In this work, I have set out to investigate whether by identifying genes contributing to self-renewal of Group 3 MB cells, we can characterize a population of cells responsible for therapy evasion and subsequent tumor relapse. Initially, we have adapted the existing COG (Children’s Oncology Group) protocol for children with newly diagnosed high-risk MB for treatment of immuno-deficient mice intracranially xenografted with human MB cells. Cell populations recovered separately from brains and spines mice during the course of tumor development and therapy were comprehensively profiled for gene expression analysis, stem cell and molecular features to generate a global, comparative profile of MB cells through therapy. Additionally, we have investigated therapeutic potential of small molecules targeting BMI1, a known self-renewal regulating gene. In the setting of recurrent Group 3 MB, pharmacological inhibition of BMI1, led to a remarkable decrease in cell proliferation and self-renewal in vitro as well as reduction of local and spinal metastatic disease in vivo. Finally, by combining the established therapy-adapted patient-derived xenograft mouse model and BMI1 inhibitor, PTC-596, we have demonstrated an additive effect of two modalities and provided the pre-clinical data for the upcoming Phase I trial. Biological investigations into the drivers of MB recurrence will lead to development of new therapeutic options for children who are frequently limited to palliation. Clinically relevant mouse models of MB recurrence can serve as platforms for pre-clinical testing and validation of new treatments aimed to provide therapeutic intervention rather than palliation. / Thesis / Doctor of Philosophy (PhD) / Medulloblastoma is the most common type of brain cancer that affects children. Out of the four main subgroups of medulloblastoma, tumors in Groups 3 and 4 are the most aggressive and are associated with a low overall survival in children diagnosed with this type of brain cancer. These two subtypes of medulloblastoma also account for the largest number of patients in which gold standard therapies fail and no additional therapies are available. Several studies have shown the existence of few cells within the tumor that alone can drive tumor growth. The aggressive behavior of these cells has in part been attributed to dysregulation of genes involved in cell replication and division. Further studies that will focus on understanding the significance of genes that regulate cell growth and replication can help discover a population of cells that is capable of evading therapy and contribute to tumor relapse. The identification and characterization of such population can lead to development of novel treatments for the children affected with aggressive medulloblastoma. In my thesis, I have developed a mouse model that replicates the aggressive therapy given to the medulloblastoma patients in order to study cells capable of escaping the harsh treatment and drive tumor comeback. Next, by profiling the gene expression and functional attributes of those cells, we identified genes that contribute to regulation of cell division and growth. The effects of both increasing and decreasing the activity of those genes were then tested in cells grown in the dish. Subsequently, the most promising results were verified in the established mouse models. The main objective of my thesis was to discover new opportunities in treatments the most aggressive type of brain cancer affecting children, and thus not only improve the quality of treatment but also the overall survival of patients with medulloblastoma.

Cancer is the most fatal disease after cardiovascular disease with over 8.2 million deaths worldwide each year. Ever since the serendipitous discovery of mustard gas as an anti-cancer therapeutic in the 1940s, serious efforts have been put into discovering more chemotherapies. Chemotherapies can be categorized into different groups such as alkylating agents (cisplatin, cyclophosphamide), antimetabolites (5-fluorouracil, Ara-C) and mitotic inhibitors (taxanes, vinca alkoids) among others. While chemotherapies have proven to kill cancer cells by targeting cell division processes, over time, tumor cells can adapt and become resistant to these drugs. With a growing understanding of cell signaling networks, targeted therapies are being developed to overcome the issue of chemotherapy resistance. Targeted therapies are highly specific molecules that bind to a specific cellular protein or molecule and block signaling networks associated with biological processes. One of the most frequently dysregulated receptor systems in cancers is the receptor tyrosine kinase family with ErbB being one of the most studied receptors families. ErbB or HER receptors consists of four structurally related receptor tyrosine kinases namely, EGFR/ErbB1, HER2/ErbB2, HER3/ErbB3 and HER4/ErbB4. The ErbB family of receptors plays a major role in morphogenesis of the human body as well as various cellular responses such as cell growth, differentiation and proliferation. Overexpression and dysregulation of these receptors, particularly EGFR and HER2, have been linked to a number of cancers such as breast cancer, gastric cancer, ovarian cancer and non-small cell lung cancer, to name a few. One of the most successful therapies against ErbB related cancers have been targeted therapies. Targeted therapies for ErbB related cancers are of two kinds: (i) Small molecule tyrosine kinase inhibitors (such as erlotinib and gefitinib against EGFR) and, (ii) Monoclonal antibodies (such as trastuzumab against HER2 and cetuximab against EGFR). These drugs function either by inhibiting the kinase activity of the receptor and preventing phosophorylation of tyrosine residues, or binding to some other site on the extracellular domain of the receptor and preventing ligand binding and heterodimerization of ErbB monomers. These drugs have proven to have limited efficacy as monotherapy, but are more effective in combination with standard chemotherapies. However, tumor cells can adapt their signaling networks developing resistance to targeted therapies over the course of treatment and lead to cancer progression. While overexpression and dysfunction of EGFR and HER2 are implicated in most ErbB driven cancers, recent studies have found HER3 playing a pivotal role in inducing resistance to EGFR and HER2 targeted therapies in various cancers and has been found to be the most sensitive node in driving the PI3K pathway leading to tumorigenesis. Thus, there is an urgent need to develop drugs targeted against HER3 and bring them into the clinic. Since HER3 lacks kinase activity, only monoclonal antibodies can be developed against it. Currently, there are a number of molecules in clinical development that target HER3. For example, patritumab and MM-121 are humanized monoclonal antibodies that target the extracellular domain of HER3 receptor and leads to inhibition of HER3-PI3K signaling followed by rapid internalization of the receptor. MM-111 and MM-141, two different bispecific monoclonal antibodies that bind to HER2, HER3 and IGFR-1, HER3, respectively, are currently in clinical development. HER3 inhibitors provide hope to effectively overcome HER3 induced tumor resistance and successfully treat several ErbB driven cancers. However, further development of HER3 inhibitors is necessary by taking strategic approaches. One of these approaches it the utilization of systems biology, a branch of biology that involves computational and mathematical modeling of complex biological systems with the aim of discovering emergent properties of biological systems. Systems biology enables researchers to get a deeper understanding of biological networks such as that of ErbB and make predictive models and test outcomes. This approach was used by Merrimack Pharmaceuticals to develop novel monoclonal antibodies against HER3. Computational outcomes were successfully validated by in vitro and in vivo experiments. Thus, this suggests that systems biology might be the future of designing and developing HER3 inhibitors that would successfully overcome HER3 resistance and cancer progression.

Les glioblastomes (GB) sont des tumeurs cérébrales invasives, résistantes et qui récidivent systématiquement malgré un traitement associant chirurgie, radio- et chimiothérapie. Ces tumeurs de très mauvais pronostic, se caractérisent par une survie médiane de 15 mois. L'agressivité des GB est notamment due à la présence d'une sous-population de cellules souches (GSC). Les GSC sont caractérisées par leurs capacités d'auto-renouvellement, d'expression de différents marqueurs souches, de multipotence et de tumorigenèse. Elles sont fortement impliquées dans la résistance et la récidive tumorale et leur ciblage pourrait améliorer le traitement des GB. Au vu de la littérature et de résultats obtenus au laboratoire, l'intégrine ß8 est apparue comme une nouvelle cible potentielle de ces GSC. Cette intégrine est décrite comme possédant un rôle majeur dans la survie et l'auto-renouvellement des cellules progénitrices neurales saines et sa surexpression est associée à une diminution de la survie des patients. Nous avons alors émis l'hypothèse que l'intégrine ß8 pourrait être impliquée dans le maintien de l'état souche des GSC. J'ai démontré au cours de ma thèse que cette intégrine est surexprimée dans des primocultures de GSC issues de résections de GB ainsi que dans des coupes de tumeurs de patients. De plus, j'ai mis en évidence que ß8 est associée à l'état souche et à des fonctionnalités propres à ces cellules, notamment leur auto-renouvellement, leur viabilité, leur migration et leur radiorésistance. En inhibant sélectivement ß8 dans nos primocultures de GSC par si/shRNA, j'ai en effet observé in vitro une diminution de la formation de neurosphères et de la migration cellulaire ainsi qu'une augmentation de la différentiation et de la mort cellulaire, cette dernière étant potentialisée après irradiation. Enfin, in vivo, j'ai mis en évidence que l'inhibition de ß8 se traduit par une diminution de la tumorigenèse et une augmentation de la survie des souris. En conclusion, mes résultats de thèse permettent d'identifier l'intégrine ß8 comme une protéine membranaire nécessaire au maintien de l'état souche dans les GSC mais surtout comme une potentielle cible thérapeutique radiosensibilisante dans les GB. / Glioblastomas (GB) are invasive, resistant and recurrent brain tumors (median overall survival of 15 months) despite standard treatment including surgical resection, radio- and chemotherapy. This tumor aggressiveness could partly be explained by the presence into the tumor of Glioblastoma-Stem like Cells (GSC), characterized by their ability to self-renew, their higher expression of specific GSC markers, their multipotent aptitude and their high tumorigenic potential. They are strongly involved in tumor resistance and recurrence and their targeting could improve GB treatment. Regarding current literature but also transcriptomic results obtained in our lab, a specific ß8 integrin emerged as a potential selective target in GSC. We then hypothesized that ß8 integrin could be involved in stemness maintenance of GSC. I first demonstrated, during my doctoral thesis, that ß8 is overexpressed in primocultures of GSC isolated from patients resections and also in human GB samples. Moreover, I showed that this integrin could be associated with stemness and features unique to these cells, including self-renewal ability, viability, migration and radioresistance. Indeed, the selective inhibition of ß8 in GSC by si/shRNA resulted in vitro in a decrease of neurosphere formation and migration, associated with an increase of differentiation patterns and cell death, this one being potentiated after irradiation. Finally, in vivo, I showed that ß8 inhibition decreased tumorigenesis and increased mice survival. In conclusion, my doctoral results allow to identify ß8 integrin as a membrane protein essential for stemness maintenance of GSC but mostly as a new potential radiosensitizing therapeutic target for GB.

No description available.

Yes / Currently, out of the 82 US FDA-approved targeted therapies for adult cancer treatments, only three are approved for use in children irrespective of their genomic status. Apart from leukemia, only a handful of genomic-based trials involving children with solid tumors are ongoing. Emerging genomic data for pediatric solid tumors may facilitate the development of precision medicine in pediatric patients. Here, we provide an up-to-date review of all reported genomic aberrations in the eight most common pediatric solid tumors with whole-exome sequencing or whole-genome sequencing data (from cBioPortal database, Pediatric Cancer Genome Project, Therapeutically Applicable Research to Generate Effective Treatments) and additional non-whole-exome sequencing studies. Potential druggable events are highlighted and discussed so as to facilitate preclinical and clinical research in this area. / Seed Grant of Strategic Research Theme for Cancer, The University of Hong Kong of AKSC. VWY Lui is funded by the Research Grant Council, Hong Kong (#17114814, #17121616, General Research Fund; T12–401/13-R, Theme-based Research Scheme), and the Start-up Fund, School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong. W Piao is funded by the Faculty Postdoctoral Fellowship Scheme, Faculty of Medicine, the Chinese University of Hong Kong.

Acute myeloid leukemia (AML) is a life-threatening malignant disorder with dismal prognosis. AML is characterized by frequent genetic changes involving tyrosine kinases, normally acting as important mediators in many basic cellular processes. Due to the overexpression and frequent mutations of the FMS-like receptor tyrosine kinase 3 (FLT3) in AML, this tyrosine kinase receptor has become one of the most sought after targets in AML drug development. In this thesis, we have used a combination of high-throughput screens, direct target interaction assays and sequential cellular screens, including primary patient samples, as an approach to discover new targeted therapies. Gefitinib, a previously known inhibitor of epidermal growth factor receptor and the two novel tyrosine kinase inhibitors AKN-032 and AKN-028, have been identified as compounds with cytotoxic activity in AML. AKN-028 is a potent inhibitor of FLT3 with an IC50 value of 6 nM in an enzyme assay, but also displaying in vitro activity in a variety of primary AML samples, irrespective of FLT3 mutation status or quantitative FLT3 expression. AKN-028 shows a sequence dependent in vitro synergy when combined with standard cytotoxic agents cytarabine or daunorubicin, with better efficacy when cells are exposed to standard chemotherapy simultaneously or for 24 hours prior to adding AKN-028. Antagonism is observed when cells are pre-treated with AKN-028, possibly explained by the cell cycle arrest induced by the compound. In vivo cytotoxic activity and good oral bioavailability have made AKN-028 a candidate drug for clinical studies and the compound is presently investigated in an international two-part multicenter phase I/II study. Results from microarray studies performed to further elucidate the mechanism of action of AKN-028, revealed significantly altered gene expression induced by AKN-028 in both AML cell lines and in primary AML cells, with an enrichment of the Myc pathway among the downregulated genes. Furthermore, tyrosine kinase activity profiling shows a dose-dependent kinase inhibition by AKN-028 in all AML samples tested. Interestingly, cells with a high overall kinase activity were more sensitive to AKN-028. Provided conformation in a larger set of samples, kinase activity profiling may give useful information in individualizing treatment of patients with AML.

Le mélanome cutané est le cancer de la peau le plus agressif de par sa grande plasticité phénotypique, son fort caractère métastatique et sa résistance aux traitements. L’émergence d’inhibiteurs ciblant la forme mutée de la kinase BRAF (BRAF V600E) a produit des réponses thérapeutiques spectaculaires, malheureusement suivies par l’apparition rapide de résistances secondaires très agressives. La compréhension des mécanismes cellulaires et moléculaires impliqués dans ces résistances constitue donc un prérequis indispensable à l’amélioration de ces thérapies ciblées. A côté des altérations intrinsèques au mélanome, les interactions entre les cellules malignes et leur microenvironnement favorisent la survie tumorale et contribuent à la résistance aux thérapies. En particulier, la matrice extracellulaire (MEC), qui constitue un réseau dynamique de macromolécules de composition et de propriétés physico-chimiques variables, influence l’architecture des tissus tumoraux, l’invasion et la réponse aux traitements. De façon importante, l’acquisition par les cellules de mélanome d’un phénotype mésenchymateux invasif a été décrite comme un mécanisme d’échappement aux thérapies ciblant la mutation oncogénique de BRAF. Dans ce contexte, l’objectif de mon travail de thèse a été de préciser le rôle de cette signature phénotypique sur les propriétés bio-mécaniques des cellules de mélanome et la réponse aux thérapies ciblées. Dans la première partie de ma thèse, j’ai observé que les cellules résistantes présentant un phénotype invasif mésenchymateux produisent, assemblent et remodèlent une matrice ayant des propriétés mécaniques et biochimiques proches de myofibroblastes. Ce phénotype est associé à une activation de la voie YAP/TAZ et une mécano-sensibilité amplifiée. La caractérisation par spectrométrie de masse du matrisome des cellules résistantes a révélé la présence abondante de protéines matricielles comme la Fibronectine, le Collagène 1(I) et la THBS1 mais également de protéines de réticulation du collagène comme LOXL2 et TGM2. Nos données montrent aussi que ces modifications sont conférées de novo par un traitement aux inhibiteurs de BRAF ou de MEK dans des cellules de mélanome mutées BRAF in vitro, et que chez la souris le traitement au Vémurafénib de cellules de mélanome xénogreffées induit l’assemblage de fibres de collagène associé à une rigidification tumorale. Finalement, j’ai pu montrer que la matrice produite par les cellules mésenchymales résistantes protège les cellules de mélanome naïves des effets anti-prolifératifs liées à l’inhibition de BRAF ou MEK. Dans une deuxième partie de ma thèse, je me suis intéressée à la protéine de réticulation du collagène de la famille des lysyl oxidases (LOX), LOXL2 exprimée par les cellules mésenchymales résistantes. Nos analyses bioinformatiques et biochimiques montrent que l'expression de cette enzyme est fortement associée à la signature invasive MITFlow AXLhigh des mélanomes. En utilisant des approches d'interférence à ARN, j'ai aussi montré que la suppression de LOXL2 dans les cellules de mélanome invasif diminue la migration cellulaire et augmente la prolifération cellulaire in vitro et in vivo, suggérant un rôle de LOXL2 dans la transition phénotypique du mélanome. Dans l’ensemble, mes travaux de thèse révèlent un rôle paradoxal de l’inhibition de la voie MAPK qui induit des changements du phénotype tumoral associés à la production autonome par la cellule maligne d’une MEC pathologique capable d'altérer le comportement cellulaire et la réponse au traitement. Cet environnement matriciel 'sanctuaire', associée à une intense hétérogénéité tumorale, pourrait jouer un rôle majeur dans le développement et l'émergence des résistances thérapeutiques du mélanome. Ces résultats permettent une meilleure compréhension du rôle de la MEC du mélanome et devraient proposer de nouvelles pistes pour améliorer les traitements. / Cutaneous melanoma remains one of the most challenging and difficult cancers to treat because of its high plasticity, metastatic potential and resistance to treatment. New therapies targeting oncogenic BRAFV600E mutation have shown remarkable clinical efficacy. However, drug resistance invariably develops. Thus, the need for improving existing therapies remains critical. Recent studies have indicated that tumor resistance arises from the tumor microenvironment in which the extracellular matrix (ECM) is a determinant factor. Here, we found that BRAF inhibitor (BRAFi)-resistant melanoma cells, but not BRAFi-sensitive cells display an increased mechanosensitivity associated with a capacity to produce and remodel a 3D ECM displaying increased levels of matrix proteins such as fibronectin (FN) and collagen fibers. Interestingly, our results show that this 3D ECM is able to protect therapy-sensitive cells from the anti-proliferative effects of MAPKi. In addition, short exposures of naive melanoma cells to MAPKi augment matrix proteins production and assembly in vitro and in vivo. This 3D ECM also promotes drug tolerance within BRAFi sensitive cells. In conclusion, our results suggest that a subset of resistance to MAPK targeted therapies is associated with the production by melanoma cells of a pathological fibrotic matrisome that may affect cell behavior and therapeutic response.

Les analyses moléculaires et la classification des adénocarcinomes bronchiques ont conduit au développement de thérapies ciblées sélectives visant à améliorer le contrôle de la maladie et la survie des patients. ALK (anaplastic lymphoma kinase) est un récepteur tyrosine kinase de la famille des récepteurs de l'insuline. Des réarrangements chromosomiques impliquant le domaine kinase d’ALK sont présents dans environ 3 à 6% des patients atteints d'un adénocarcinome bronchique. La protéine de fusion provoque une activation du domaine kinase de manière constitutive et indépendante du ligand. Lorlatinib est un inhibiteur d’ALK de troisième génération avec une efficacité et une sélectivité optimale, ainsi qu’une pénétration élevée vers le système nerveux central. Lorlatinib peut vaincre la résistance induite par plus de 16 mutations secondaires dans le domaine kinase d’ALK acquises lors de la progression aux ALK TKI de première et deuxième générations. Le traitement par lorlatinib est donc efficace chez les patients préalablement traités par un ALK TKI de première ou deuxième génération, et est actuellement approuvé pour cette indication. Le spectre complet de mécanismes de résistance au lorlatinib chez les patients reste à élucider. Il a récemment été rapporté que l'acquisition séquentielle de deux mutations ou plus dans le domaine kinase, également appelées mutations composées, est responsable de la progression de la maladie chez environ 35% des patients traités par le lorlatinib, principalement en altérant sa liaison au domaine kinase d’ALK. Cependant, l’effet de ces mutations sur la sensibilité aux différents inhibiteurs d’ALK peut varier, et les autres mécanismes de résistance survenant chez la plupart des patients restent inconnus. Mon travail de thèse avait pour but d’explorer la résistance au lorlatinib chez des patients atteints d'un cancer du poumon ALK réarrangé par la mise en œuvre de biopsies spatiales et temporelles et le développement de modèles dérivés de patients. Dans le cadre de l’étude institutionnelle MATCH-R (NCT02517892), nous avons effectué un séquençage à haut débit de l’exome, de l’ARN et ciblé, ainsi qu’un séquençage des ctDNA afin d’identifier les mécanismes de résistance. Nous avons établi des lignées cellulaires dérivées de patients et caractérisé de nouveaux mécanismes de résistance et identifiés de nouvelles stratégies thérapeutiques in vitro et in vivo. Nous avons identifié trois mécanismes de résistance chez quatre patients avec des biopsies appariées. Nous avons étudié l'induction de la transition épithélio-mésenchymateuse (EMT) par l'activation de SRC dans une lignée cellulaire, dérivée d’un patient, exposée au lorlatinib. Les cellules mésenchymateuses étaient sensibles à l’inhibition combinée de SRC et d'ALK, montrant que même en présence d'un phénotype agressif, des stratégies de combinaison peuvent surmonter la résistance aux ALK TKI. Nous avons identifié deux nouvelles mutations composées du domaine kinase d’ALK, F1174L / G1202R, C1156Y / G1269A survenues chez deux patients traités par le lorlatinib. Nous avons développé des modèles de cellules Ba / F3 exprimant les mutations simples et composées pour étudier leur effet sur la résistance au lorlatinib. Enfin, nous avons caractérisé un nouveau mécanisme de résistance provoqué par la perte de fonction de NF2 au moment de la progression du lorlatinib par l’utilisation de PDX et de lignées cellulaires dérivées de patients, et par CRISPR / CAS9 knock-out de NF2. Nous avons constaté que l'activation de mTOR par la perte de fonction de NF2 provoquait la résistance au lorlatinib et qu'elle pouvait être surmontée par le traitement avec des inhibiteurs de mTOR.Cette étude montre que les mécanismes de résistance au lorlatinib sont plus divers et complexes que prévu. Nos résultats démontrent également comment les études longitudinales de la dynamique tumorale permettent de déchiffrer la résistance aux TKI et d'identifier des stratégies thérapeutiques. / The molecular study and classification of lung adenocarcinomas has led to the development of selective targeted therapies aiming to improve disease control and survival in patients. The anaplastic lymphoma kinase (ALK) is a tyrosine kinase receptor from the insulin tyrosine kinase receptor family, with a physiologic role in neural development. Gene rearrangements involving the ALK kinase domain occur in ~3-6% of patients with lung adenocarcinoma. The fusion protein dimerizes leading to transactivation of the ALK kinase domain in a ligand-independent and constitutive manner. Lorlatinib is a third generation ALK inhibitor with high potency and selectivity for this kinase in vitro and in vivo, and elevated penetrance in the central nervous system. Lorlatinib can overcome resistance mediated by over 16 secondary kinase domain mutations occurring in 13 residues upon progression to first - and second - generation ALK TKI. In addition, treatment with lorlatinib is effective for patients who have been previously treated with a first and a second generation or a second generation ALK TKI upfront and is currently approved for this indication. The full spectrum of biological mechanisms driving lorlatinib resistance in patients remains to be elucidated. It has been recently reported that the sequential acquisition of two or more mutations in the kinase domain, also referred as compound mutations, is responsible for disease progression in about 35% of patients treated with lorlatinib, mainly by impairing its binding to the ALK kinase domain. However, the effect of these compound mutations on the sensitivity to the repertoire of ALK inhibitors can vary, and other resistance mechanisms occurring in most patients are unknown. My PhD thesis aimed at exploring resistance to lorlatinib in patients with ALK-rearranged lung cancer through spatial and temporal tumor biopsies and development of patient-derived models. Within the institutional MATCH-R study (NCT02517892), we performed high-throughput whole exome, RNA and targeted next-generation sequencing, together with plasma sequencing to identify putative genomic and bypass mechanisms of resistance. We developed patient-derived cell lines and characterized novel mechanisms of resistance and personalized treatment strategies in vitro and in vivo. We characterized three mechanisms of resistance in four patients with paired biopsies. We studied the induction of epithelial-mesenchymal transition (EMT) by SRC activation in a patient-derived cell line exposed to lorlatinib. Mesenchymal cells were sensitive to combined SRC and ALK co-inhibition, showing that even in the presence of an aggressive and challenging phenotype, combination strategies can overcome ALK resistance. We identified two novel ALK kinase domain compound mutations, F1174L/G1202R, C1156Y/G1269A, occurring in two patients treated with lorlatinib. We developed Ba/F3 cell models harboring single and compound mutations to study the differential effect of these mutations on lorlatinib resistance. Finally, we characterized a novel mechanism of resistance caused by NF2 loss of function at the time of lorlatinib progression through the development of patients derived PDX and cell lines, and in vitro validation of NF2 knock-out with CRISPR/CAS9 gene editing. Downstream activation of mTOR was found to drive lorlatinib resistance by NF2 loss of function and was overcome by providing treatment with mTOR inhibitors.This study shows that mechanisms of resistance to lorlatinib are more diverse and complex than anticipated. Our findings also emphasize how longitudinal studies of tumor dynamics allow deciphering TKI resistance and identifying reversing strategies.

Prostate cancer (PCa) bone metastases have been reported in ~90% of patients with advanced disease. Bone metastases disrupt tissue homeostasis and weaken the skeleton, resulting in an increased risk of bone fractures and morbidity. Specifically, PCa cells disrupt the crosstalk between critical cells within the tumor/bone microenvironment (osteoblasts, osteoclasts, and immune cells), and utilize this effector-rich environment for cancer survival and growth. Therefore, a key therapeutic objective in malignant skeletal disease management is to eliminate tumors while restoring bone homeostasis. Current treatments include palliative radiotherapy, chemotherapy, or anti-RANK treatments, all of which have considerable side effects such as osteonecrosis of the jaw or enhanced tumor invasion. There remains a critical gap in therapies than can reduce tumor burden and simultaneously restore bone homeostasis. To address this gap, our work explores emerging gene therapy approaches for treating skeletal malignancies by utilizing multifunctional cytokine-based agents that can simultaneously combat tumor growth and promote bone regeneration.<div><br></div><div>We hypothesize that rationally designed cytokine-based gene therapies that can be secreted from skeletal muscle and targeted to the bone/tumor microenvironment, could effectively reduce tumor growth and restore bone cell homeostasis. To test this hypothesis, we adopted two strategies: 1) a second-generation targeted IL-27 cytokine, and 2) a de novodesign of a cytokine-like therapeutic agent (Propeptide) that includes anti-tumorigenic and pro-osteogenic domains. Both strategies share modules with overlapping therapeutic functions, rendering them complementary in their therapeutic application. In this work, we examined the proof of principle for propeptide gene therapy in muscle cells (in vitro models) and assessed the therapeutic efficacy of our cytokine-based biologics in reducing prostate tumor growth and rebalancing bone cell proliferation and differentiation. Our studies resulted in a propeptide construct representative of a cytokine structure comprised of a bundle of helices that we were able to express in cells. Additionally, our work demonstrated the targeting and anti-tumor efficacy of our therapeutic cytokines in cancer and bone cell models. Ultimately, this will provide the framework for innovative peptide and cytokine-based therapeutics that target and treat both the tumor metastases and bone. This approach will facilitate improvement of morbidity and quality of life of prostate cancer patients with bone metastases and could be applicable to other diseases with bone/tumor pathologies. <br><div><br></div></div>

No description available.