Investigating the role of Microphthalmia-Associated transcription Factor M in melanoma development and drug resistance

Malignant melanoma is an aggressive cancer, and the prognosis is poor for patients with advanced disease. Currently, melanoma treatment consists of immunotherapy and therapy targeting the MAPK pathway. However, many patients do not respond, or develop treatment resistance. Identifying factors that contribute to melanoma initiation and/or drug resistance is therefore of interest. MITF-M is a master transcription factor that plays a dominant role in both melanocyte and melanoma biology, and elucidation of some of the aspects of the MITF-M protein signal pathways has been the subject of this thesis. In the first thesis project, we identified MITF-M as a master regulator of vemurafenib (BRAF inhibitor)-induced drug resistance. During vemurafenib treatment of melanoma cell lines, loss of MITF-M was found to upregulate the ERBB3 receptor and its cognate ligand NRG, both having effect on sustained growth and survival. Further, we investigated the role of MITF-M and SOX10 upon growth receptors their ligands using cell panels during development of vemurafenib-induced drug resistance. We suggest that identifying SOX10 and MITF levels prior to treatment initiation may offer a tool to overcome drug resistance or prolong combinatory treatment efficacy. In our final project, the role of MITF in the context of MC1R status was investigated, as mutations in MC1R may contribute to increased risk for melanoma. Here, we developed a cell line model for studying the signaling networks that contributes to melanoma development in individual’s carrying predisposing mutations in the MC1R gene. In conclusion, we have identified novel roles of MITF-M, contributing to increased understanding of melanoma progression and vemurafenib-induced drug resistance in melanoma

Overexpression of SMPX in adult skeletal muscle does not change skeletal muscle fiber type or size

Mechanical factors such as stretch are thought to be important in the regulation of muscle phenotype. Small muscle protein X-linked (SMPX) is upregulated by stretch in skeletal muscle and has been suggested to serve both as a transcription factor and a mechanosensor, possibly giving rise to changes in both fiber size and fiber type. We have used in vivo confocal imaging to study the subcellular localization of SMPX in skeletal muscle fibers of adult rats using a SMPX-EGFP fusion protein. The fusion protein was localized predominantly in repetitive double stripes flanking the Z-disc, and was excluded from all nuclei. This localization would be consistent with SMPX being a mechanoreceptor, but not with SMPX playing a role as a transcription factor. In vivo overexpression of ectopic SMPX in skeletal muscle of adult mice gave no significant changes in fiber type distribution or cross sectional area, thus a role of SMPX in regulating muscle phenotype remains unclear

SMPX-EGFP was excluded from all myonuclei.

<p>A) C2C12 myoblasts expressing either EGFP or SMPX-EGFP stained with Hoechst 33342 to visualize nuclei. B) <i>In vitro</i> confocal image of dissected single rat EDL muscle fibers expressing EGFP or SMPX-EGFP stained with DAPI to visualize nuclei. C) Confocal images of EDL muscle fibers <i>in situ,</i> after no treatment (CON) or functional overload for 18 hours (LOAD), expressing SMPX-EGFP stained with Hoechst 33342 to visualize nuclei. Myonuclei are labeled with arrowheads. Scale bar is 10 microns.</p

Muscle fiber type and cross sectional area (CSA) in mouse EDL.

<p>Single fibers from muscles transfected with either pCMS-EGFP (left leg sham control, only expressing EGFP) or pCMS-EGFP-<i>Smpx</i> (expressing SMPX and EGFP). A) CSA with n = 226/254 cells for sham/SMPX. Values are means +/- SD. B) Fiber type composition of the same fibers as in A.</p

SMPX-EGFP was localized in bands flanking the z-disc.

<p>A) <i>In situ</i> confocal image of EGFP or SMPX-EGFP expression from an isolated EDL rat muscle in Ringer-solution. Scale bar is 10 microns. B) <i>In vitro</i> confocal image of EGFP or SMPX-EGFP stained with Alexa Fluor 680 Phalloidin (red) to visualize actin filaments. Scale bar is 10 microns (inset 1 micron).</p

Expression of SMPX plasmids, in vitro and in vivo.

<p>A) Western blot of SMPX-EGFP expression in HEK-293 cells stained with antibodies against EGFP. Lane 1: Negative control. Lane 2: EGFP only. Lane 3-6: SMPX-EGFP. B) <i>In vivo</i> fluorescence image of EDL muscle expressing pCMS-EGFP-<i>Smpx</i>. Scalebar is 500 microns. Inset: High magnification of single fibers. Scalebar is 50 microns. C) β-gal (left) and myosin heavy chain type 2A (right) stain on neighboring cross-sections from the same muscle as in B). β-gal expressing fibers marked with asterisks. Scale bar is 50 microns.</p

Dysregulation of MITF Leads to Transformation in MC1R-Defective Melanocytes

The MC1R/cAMP/MITF pathway is a key determinant for growth, differentiation, and survival of melanocytes and melanoma. MITF-M is the melanocyte-specific isoform of Microphthalmia-associated Transcription Factor (MITF) in human melanoma. Here we use two melanocyte cell lines to show that forced expression of hemagglutinin (HA) -tagged MITF-M through lentiviral transduction represents an oncogenic insult leading to consistent cell transformation of the immortalized melanocyte cell line Hermes 4C, being a melanocortin-1 receptor (MC1R) compound heterozygote, while not causing transformation of the MC1R wild type cell line Hermes 3C. The transformed HA-tagged MITF-M transduced Hermes 4C cells form colonies in soft agar and tumors in mice. Further, Hermes 4C cells display increased MITF chromatin binding, and transcriptional reprogramming consistent with an invasive melanoma phenotype. Mechanistically, forced expression of MITF-M drives the upregulation of the AXL tyrosine receptor kinase (AXL), with concomitant downregulation of phosphatase and tensin homolog (PTEN), leading to increased activation of the PI3K/AKT pathway. Treatment with AXL inhibitors reduces growth of the transformed cells by reverting AKT activation. In conclusion, we present a model system of melanoma development, driven by MITF-M in the context of MC1R loss of function, and independent of UV exposure. This model provides a basis for further studies of critical changes in the melanocyte transformation process

Atezolizumab and stereotactic body radiotherapy in patients with advanced non-small cell lung cancer: safety, clinical activity and ctDNA responses—the ComIT-1 trial

The introduction of immune checkpoint inhibitors has transformed the treatment landscape of metastatic non-small cell lung cancer. However, challenges remain to increase the fraction of patients achieving durable clinical responses to these drugs and to help monitor the treatment effect. In this phase II trial, we investigated the toxicity, systemic responses and circulating tumour DNA responses in patients (n = 21) with advanced non-small-cell lung cancer treated with atezolizumab and stereotactic body radiotherapy in the second or later line. We found the combined treatment to be safe with grade 3 toxicity reported in three patients. As the best overall response, four patients had a partial response, eight had stable disease and five had progressive disease. Median overall survival time was still not reached after a median follow-up of 26.5 months and 10/15 patients with programmed death-ligand 1 negative tumours were alive >18 months after the start of the study treatment. ctDNA was detectable at baseline in 11 patients. A rapid decline in ctDNA to <30% of baseline levels was seen in three patients, two of which were radiographic responders and one was considered clinically benefiting from therapy for almost 1 year