Evaluating Targeted And Immunomodulatory Therapies For Melanoma In A Genetically Engineered Mouse Melanoma Model

Melanoma therapy has changed rapidly due to the emergence of new therapies: MAPK-pathway targeted drugs and immunomodulatory agents. Given the relative success of these new individual drugs, this work set out to evaluate and develop effective melanoma treatments using combination therapies in a preclinical mouse melanoma models. Therapies tested include BRAF kinase inhibition in combination with: immune checkpoint inhibitors anti-CTLA4, anti-PDL1, and with the topical TLR7/8 agonist imiquimod. Drugs efficacies were tested in established melanomas in a conditional inducible mouse melanoma model based on activation of Braf and beta catenin and loss of Pten. BRAF inhibition in combination with anti-CTLA-4/anti-PD-L1 was not more effective than BRAF inhibition alone in retarding tumor growth or prolonging survival in these studies. Treatment with imiquimod significantly retarded tumor growth and increased survival. Imiquimod-treated tumors show increased macrophage infiltration, but not increased intratumoral T lymphocytes. Further work remains to identify effective, synergistic drug combinations in preclinical models

Novel prognostic factors for advanced melanoma and localized renal cell carcinoma

This study aimed to evaluate prognostic and predictive factors in melanoma and renal cell carcinoma to tailor optimal treatment and follow-up for cancer patients. Chemotherapy was the standard treatment for advanced melanoma before immune checkpoint inhibitors and targeted therapies. The median overall survival was 8.9 months (95% CI 7.5–10.4) and the five-year survival rate 13% in 146 patients who had received BOLD-IFN chemoimmunotherapy at Turku University Hospital in 1991─2010. Long-term survivors were found especially in patients without visceral metastases (five-year survival rate 28%). The Finnish Melanoma Group conducted a prospective, multicenter trial enrolling 38 patients who received TOL-IFN (temozolomide, lomustine, vincristine, and interferone-alpha) ± vemurafenib for the first-line treatment of advanced cutaneous melanoma. Elevated LDH was associated with shorter overall survival unlike asymptomatic brain metastases. Undetectable circulating tumor DNA in baseline plasma samples correlated with longer progression-free survival and baseline ctDNA levels were inversely associated with overall survival. Patients with persistent detectable ctDNA during treatment had the shortest overall survival. One-third of patients will develop disease recurrence after surgery for localized renal cell carcinoma. Tumor size, tumor grade (Fuhrman), and microvascular invasion were sufficient for the accurate prediction of metastasis-free survival in 196 patients operated for localized clear cell RCC. The three-feature prediction model was validated in an external cohort of 714 patients. It retained similar prediction accuracy as the Leibovich model (C-index 0.836 vs. 0.848, p=0.106) and had better prognostic value for long-term prediction in both cohorts In conclusion, undetectable ctDNA is a novel biomarker indicating favourable prognosis in advanced melanoma. This study suggests that patients with persistent detectable ctDNA may require more frequent monitoring of treatment response and perhaps more intensive therapy. We also introduced a three-feature prediction model for metastasis-free survival as a tool for optimizing postoperative follow-up of localized RCC patients.Edenneen melanooman ja paikallisen munuaissyövän uudet ennustetekijät Tämän väitöskirjatutkimuksen tavoitteena oli löytää uusia ennustetekijöitä, jotka voivat auttaa suunnittelemaan melanooma- ja munuaissyöpäpotilaiden yksilöllistä hoitoa ja seurantaa. Ensimmäisessä osatyössä tutkittiin solunsalpaajahoidon ja alfainterferonin (DOBC-IFN) hyötyä ennustavia tekijöitä. 146 potilasta oli saanut DOBC-IFN-hoitoa TYKS:ssä edenneen ihomelanooman vuoksi vuosina 1991–2010. Potilaiden eliniän mediaani oli 8,9 kuukautta (95 prosentin luottamusväli 7,5–10,4 kk) ja viiden vuoden kohdalla elossa olevien potilaiden osuus oli 13 prosenttia. Jopa 28 prosenttia potilaista, joilla ei ollut todettu sisäelinetäpesäkkeitä, pysyi elossa viisi vuotta. Toisessa ja kolmannessa osatyössä raportoitiin tulokset Suomen Melanoomaryhmän toteuttamasta prospektiivisesta kansallisesta monikeskustutkimuksesta, jossa annettiin 38:lle edennyttä ihomelanoomaa sairastavalle potilaalle solunsalpaajien, alfainterferonin (TOL-IFN) ja vemurafenibin yhdistelmähoitoa. Korkea plasman laktaattidehydrogenaasipitoisuus ennusti lyhyempää elinaikaa, kun taas oireettomat aivometastaasit eivät olleet yhteydessä lyhyempään elinaikaan. Veressä kiertävä kasvain-DNA ennusti nopeampaa taudin etenemistä ja kasvain- DNA:n määrä oli kääntäen verrannollinen elinajan pituuteen. Lyhyin elinaika todettiin potilailla, joilla kasvain-DNA ei hävinnyt hoidon aikana toistetusti otetuista plasmanäytteistä. Neljännessä osatyössä osoitettiin, että syöpäkasvaimen koko, syöpäsolujen erilaistumisaste ja leviäminen hiusverisuoniin ennustavat luotettavasti etäpesäkkeiden ilmaantumista paikallisen kirkassoluisen munuaissyövän leikkauksen jälkeen. Johtopäätöksenä voidaan todeta, että veressä kiertävä kasvain-DNA ennustaa melanoomapotilaiden elinaikaa. Mikäli kiertävä kasvain-DNA ei häviä hoidon aikana, voidaan harkita hoidon tehostamista. Neljännessä osatyössä esitellyn uuden nomogrammin avulla voidaan arvioida potilaan riskiä sairastua levinneeseen munuaissyöpään ja tätä luokittelua voidaan käyttää, kun suunnitellaan potilaan seurantaa paikallisen kirkassoluisen munuaissyövän leikkauksen jälkeen

Targeting the Hsp90 Molecular Chaperone and Resistant Pathways in BRAF-mutant Melanoma

Melanoma remains the most aggressive and fatal type of skin cancer. In greater than 50% of cases, patients present with an activating BRAF mutation (BRAF+), leading to upregulated mitogen-activated protein kinase (MAPK) pathway signaling. Of these patients, 80-90% have a missense mutation at codon 600 (e.g., BRAFV600E), making the mutant form of the protein an attractive and druggable target. In 2011, the FDA approved combination therapy of BRAF+ and MEK inhibitors (BRAFi/MEKi), like vemurafenib and cobimetinib (Ve/Cb), for use in unresectable late-stage melanoma patients, drastically changing treatment options and initial outcomes. Still, the majority of patients become refractory to BRAFi/MEKi within the first year of treatment. The lack of treatment durability underscores the need for novel therapeutic strategies and drug candidate development, such as the utilization of molecular chaperone inhibitors. The 90-kDa heat shock protein (Hsp90) is a molecular chaperone and responsible for stabilizing the protein folding of “client” proteins that interact with the heterochaperone complex that Hsp90 forms with the 70-kDa heat shock protein (Hsp70) and other co-chaperones. These clients are involved in several cellular signaling pathways and processes, highlighting the significance of chaperone function in eukaryotic cells. Interestingly, Hsp90 expression increases several-fold in cancer cells to compensate for cellular stress and client protein dependence on chaperone function. To-date 18 small molecule Hsp90 inhibitors (Hsp90i) entered clinical trials, of which 94.4% target the N-terminus (NT-Hsp90i) but failed to get FDA approval. The NT-Hsp90i are effective and xvi potent, but pan-inhibitors of all four Hsp90 isoforms. In clinical trials, patients require dose-escalation of NT-Hsp90i to reach a therapeutic effect, ultimately leading to dose-limiting toxicities (DTL). Studies suggest a link between DTLs and the activation of the heat shock response (HSR), especially the cytoprotective role of Hsp70. Previously, our lab, with collaborators, developed novel C-terminal Hsp90i (CT-Hsp90i) and showed efficacy in several cancer models in vitro and in vivo while mitigating the HSR, suggesting a decreased toxicity profile compared to NT-Hsp90i. For this dissertation, I researched therapeutic resistance mechanisms in BRAF+ melanoma through various preclinical in vitro studies that targeted Hsp90. Specifically, I tested the hypothesis that several resistance-promoting processes require Hsp90 function and, therefore, could be targeted with an Hsp90i to simultaneously knockdown resistance pathways and oncogenic processes. First, I showed effective melanoma cell death using the CT-Hsp90i KU758 at potent micromolar concentrations (e.g., IC50 = 0.36 – 0.43 micromolar). Next, I demonstrated robust synergy (e.g., CI<0.5) of KU758 when combined with either a BRAFi or MEKi to target two resistance pathways effectively (e.g., MAPK/Erk and PI3K/Akt), significantly mitigate melanocyte migration, and downregulate key Hsps involved in HSR activation. Finally, I accessed publicly available genomic data via the National Cancer Institute and The Cancer Genome Atlas (TCGA) program to identify additional genes of interest in BRAF+ melanomas. Using a clustered heatmap of RNA expression data, I distinguished genes of interest based on common expression alterations amongst a subset of BRAF+ melanoma patients to provide a genetic perspective in the context of Hsp90i use in melanoma patients. Collectively, this work reviews the use of and development of several small xvii molecule inhibitors in melanomas (e.g., BRAFi/MEKi and Hsp90i), identifies a novel and effective KU758-combination approach in BRAF+ melanomas, and gives insight into future therapeutic directions based on various translational and genetic signatures in these difficult-to-treat tumors.PHDPharmacologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/163262/1/jaquesan_1.pd

Advances and Novel Treatment Options in Metastatic Melanoma

The book presents several studies reporting advances on melanoma pathogenesis, diagnosis and therapy. It represents a milestone on the state of the art, updated at 2021, and also presents the current knowledge on the future developments in melanoma field

Cutaneous Melanoma Classification: The Importance of High-Throughput Genomic Technologies

Cutaneous melanoma is an aggressive tumor responsible for 90% of mortality related to skin cancer. In the recent years, the discovery of driving mutations in melanoma has led to better treatment approaches. The last decade has seen a genomic revolution in the field of cancer. Such genomic revolution has led to the production of an unprecedented mole of data. High-throughput genomic technologies have facilitated the genomic, transcriptomic and epigenomic profiling of several cancers, including melanoma. Nevertheless, there are a number of newer genomic technologies that have not yet been employed in large studies. In this article we describe the current classification of cutaneous melanoma, we review the current knowledge of the main genetic alterations of cutaneous melanoma and their related impact on targeted therapies, and we describe the most recent highthroughput genomic technologies, highlighting their advantages and disadvantages. We hope that the current review will also help scientists to identify the most suitable technology to address melanoma-related relevant questions. The translation of this knowledge and all actual advancements into the clinical practice will be helpful in better defining the different molecular subsets of melanoma patients and provide new tools to address relevant questions on disease management. Genomic technologies might indeed allow to better predict the biological - and, subsequently, clinical - behavior for each subset of melanoma patients as well as to even identify all molecular changes in tumor cell populations during disease evolution toward a real achievement of a personalized medicine

Molecular Subtypes of Melanoma. Biological and Clinical Significance.

Cutaneous malignant melanoma (CMM) is the most lethal form of skin cancer and its incidence has increased faster than that of any other cancer, rendering it a major public health problem worldwide. High-throughput screenings have opened the door to a new scientific world, which enables molecular-based characterization of large cancer cohort collections. The aim of the research presented in this thesis was to explore the molecular landscapes of melanoma tumors on a genomic and transcriptomic level and subsequently correlate certain molecular features with patient survival, treatment response and tumor evolutionary patterns. In Paper I, it was concluded that metastatic melanoma could be divided into transcriptomic subtypes (gene expression (GEX) phenotypes) possessing diverse biological and clinical features. Patients harboring melanomas infiltrated by immune cells, i.e. the high-immune subtype, showed a superior survival, whereas highly proliferative melanomas, i.e. the proliferative subtype, was correlated to a poor survival outcome and resistance to targeted therapies. Moreover, it was also shown that, irrespectively of the GEX phenotypes, melanomas could be divided into genomic subtypes based on genetic aberrations in the mitogen-activated protein kinase (MAPK) signaling pathway. In Paper II, it was found that mutations in the tumor suppressor gene neurofibromin 1 (NF1), was linked to inferior survival. Today, it is well accepted that most tumors possess some level of intratumor heterogeneity (ITH), i.e. subclonality, influencing disease progression. In Papers III and IV, the evolutionary aspects of melanoma were considered by analyzing ITH, as well as disease progression on a molecular basis. When analyzing multiple metastatic lesions from individual patients, we found that most tumors were genetically different, with a common stem of genetic aberrations and the addition of new “private” ones, thus pointing to continued evolution during progression. Moreover, the GEX proliferative phenotype appeared to be correlated to a later disease course. From multiregional biopsies from single tumors, it was found that mutations in the MAPK signaling pathway appeared to be early events in tumorigenesis. Heterogeneous somatic mutations were found in the range of 3-38%, thus highlighting different levels of subclonality in melanoma. A high degree of mutational heterogeneity was associated with a more aggressive disease progression. In conclusion, melanoma is a complex molecular disease that can be characterized by genomic and transcriptomic signatures with clinical implications. However, a single biopsy might not reflect the true tumor complexity, and subclonality may be one reason behind resistance development