Remeniuk, Bethany

Abstract One of the most significant unmet clinical challenges in paediatric oncology is the development of novel therapeutic strategies for recurrent medulloblastoma (R-MB). MYC-driven MBs are defined as classically cold tumours with a low incidence of infiltrating immune cells, resulting in a therapeutic challenge. The identification of cell-cell interactions between tumour and immune cells may provide insight into critical intercellular communications and manipulations occurring within the tumour immune microenvironment (TME). We hypothesised that the key cell-cell interactions in MYC-driven primary and recurrent MB may reveal dominant immune-suppressive mechanisms and uncover targetable therapeutic vulnerabilities. Paired primary-recurrent bulk RNA-sequencing data, confirmed myeloid cells as the most infiltrating immune cell type in group3-MB and group4-MB. Comprehensive spatial phenotypic and cell-cell communication analyses corroborated this discovery, validating an increased incidence of macrophages in the matched-recurrent tumours. Subsequently, we used innovative algorithms for 10X MB single-cell data to predict interactions between tumour-cell ligands and immune-cell receptors within the TME; macrophages emerged as the core immune-cells involved in interactions throughout the TMEs, with the most significant ligand-receptor interaction and inflammatory response between MIF and CD74. In-depth immunohistochemistry analyses of primary and recurrent group3 and group4 tumours, and exhaustive tissue microarrays demonstrated expression of both CD74 and MIF, with limited expression of CD74 within the brain. To investigate the therapeutic potential of CD74, we developed recurrent, immune competent MYC-driven medulloblastoma mouse models. Comprehensive deconvolution analysis confirmed the TME integrity of our models to mirror that of the human disease. Locoregional delivery and repeat dosing of a bioactive-CD74 peptide demonstrated complete tumour clearance in our immune-competent mouse models of primary and recurrent MB, demonstrating the significant therapeutic potential of targeting the CD74-MIF axis in MYC-driven primary and recurrent MB. Key cellular interactions and therapeutic vulnerabilities within the tumour microenvironment of MYC-driven medulloblastoma (MB) have been identified, highlighting the CD74-MIF axis as a target for next-generation immunotherapies.

Abstract Background Relapsed medulloblastoma remains a significant therapeutic challenge as it is near universally fatal. The tumor microenvironment of medulloblastoma plays a critical role in tumor progression, influencing tumor growth, immune evasion, and therapeutic resistance. We hypothesized that defining tumor-immune interactions in diagnostic and relapsed medulloblastoma may uncover mechanisms of immune evasion and identify novel therapeutic targets. Methods We analyzed paired primary and recurrent RNA-sequencing data from 140 medulloblastoma patients to profile immune cell composition and validate spatial relationships within the TME. To identify key tumor-immune interactions, we developed a novel algorithm to detect receptor-ligand pairs using single-cell RNA-sequencing data. These interactions were validated across RNA and proteomic datasets. Their functional significance was empirically demonstrated in newly developed immunocompetent models of recurrent medulloblastoma that closely recapitulate the human disease. Results We observed a shift toward a heightened immunosuppressive TME at relapse. Using our algorithm, we identified biologically significant receptor-ligand interactions, most notably MIF-CD74, constitutively expressed at RNA and protein levels across medulloblastoma subgroups, at diagnosis and relapse. Disrupting MIF-CD74 interactions led to significant alterations in the tumor microenvironment, highlighting its functional significance. Conclusions Our multifaceted approach identified key tumor-immune interactions in medulloblastoma. Among these, MIF-CD74 was validated as a targetable interaction, demonstrating the utility of our integrative approach for identifying novel therapeutic targets across multiple tumor types.

Background Acute and persistent post-traumatic headache are often debilitating consequences of traumatic brain injury. Underlying physiological mechanisms of post-traumatic headache and its persistence remain unknown, and there are currently no approved therapies for these conditions. Post-traumatic headache often presents with a migraine-like phenotype. As calcitonin-gene related peptide promotes migraine headache, we explored the efficacy and timing of intervention with an anti- calcitonin-gene related peptide monoclonal antibody in novel preclinical models of acute post-traumatic headache and persistent post-traumatic headache following a mild traumatic brain injury event in mice. Methods Male, C57Bl/6 J mice received a sham procedure or mild traumatic brain injury resulting from a weight drop that allowed free head rotation while under minimal anesthesia. Periorbital and hindpaw tactile stimulation were used to assess mild traumatic brain injury-induced cutaneous allodynia. Two weeks after the injury, mice were challenged with stress, a common aggravator of migraine and post-traumatic headache, by exposure to bright lights (i.e. bright light stress) and cutaneous allodynia was measured hourly for 5 hours. A murine anti- calcitonin-gene related peptide monoclonal antibody was administered after mild traumatic brain injury at different time points to allow evaluation of the consequences of either early and sustained calcitonin-gene related peptide sequestration or late administration only prior to bright light stress. Results Mice with mild traumatic brain injury, but not a sham procedure, exhibited both periorbital and hindpaw cutaneous allodynia that resolved by post-injury day 13. Following resolution of injury-induced cutaneous allodynia, exposure to bright light stress re-instated periorbital and hindpaw cutaneous allodynia in injured, but not sham mice. Repeated administration of anti-calcitonin-gene related peptide monoclonal antibody at 2 hours, 7 and 14 days post mild traumatic brain injury significantly attenuated the expression of cutaneous allodynia when evaluated over the 14-day post injury time course and also prevented bright light stress-induced cutaneous allodynia in injured mice. Administration of anti-calcitonin-gene related peptide monoclonal antibody only at 2 hours and 7 days after mild traumatic brain injury blocked injury-induced cutaneous allodynia and partially prevented bright light stress-induced cutaneous allodynia. A single administration of anti-calcitonin-gene related peptide monoclonal antibody after the resolution of the peak injury-induced cutaneous allodynia, but prior to bright light stress challenge, did not prevent bright light stress-induced cutaneous allodynia. Conclusions We used a clinically relevant mild traumatic brain injury event in mice along with a provocative stimulus as novel models of acute post-traumatic headache and persistent post-traumatic headache. Following mild traumatic brain injury, mice demonstrated transient periorbital and hindpaw cutaneous allodynia suggestive of post-traumatic headache-related pain and establishment of central sensitization. Following resolution of injury-induced cutaneous allodynia, exposure to bright light stress re-established cutaneous allodynia, suggestive of persistent post-traumatic headache-related pain. Continuous early sequestration of calcitonin-gene related peptide prevented both acute post-traumatic headache and persistent post-traumatic headache. In contrast, delayed anti-calcitonin-gene related peptide monoclonal antibody treatment following establishment of central sensitization was ineffective in preventing persistent post-traumatic headache. These observations suggest that mechanisms involving calcitonin-gene related peptide underlie the expression of acute post-traumatic headache, and drive the development of central sensitization, increasing vulnerability to headache triggers and promoting persistent post-traumatic headache. Early and continuous calcitonin-gene related peptide blockade following mild traumatic brain injury may represent a viable treatment option for post-traumatic headache and for the prevention of post-traumatic headache persistence. Abbreviations CA Cutaneous allodynia CGRP Calcitonin gene-related peptide mTBI Mild traumatic brain injury PTH Post-traumatic headache APTH Acute post-traumatic headache PPTH Persistent post-traumatic headache

Background Stress is the most commonly reported migraine trigger. Dynorphin, an endogenous opioid peptide acting preferentially at kappa opioid receptors (KORs), is a key mediator of stress responses. The aim of this study was to use an injury-free rat model of functional cephalic pain with features of migraine and medication overuse headache (MOH) to test the possible preventive benefit of KOR blockade on stress-induced cephalic pain. Methods Following sumatriptan priming to model MOH, rats were hyper-responsive to environmental stress, demonstrating delayed cephalic and extracephalic allodynia and increased levels of CGRP in the jugular blood, consistent with commonly observed clinical outcomes during migraine. Nor-binaltorphimine (nor-BNI), a long-acting KOR antagonist or CYM51317, a novel short-acting KOR antagonist, were given systemically either during sumatriptan priming or immediately before environmental stress challenge. The effects of KOR blockade in the amygdala on stress-induced allodynia was determined by administration of nor-BNI into the right or left central nucleus of the amygdala (CeA). Results KOR blockade prevented both stress-induced allodynia and increased plasma CGRP. Stress increased dynorphin content and phosphorylated KOR in both the left and right CeA in sumatriptan-primed rats. However, KOR blockade only in the right CeA prevented stress-induced cephalic allodynia as well as extracephalic allodynia, measured in either the right or left hindpaws. U69,593, a KOR agonist, given into the right, but not the left, CeA, produced allodynia selectively in sumatriptan-primed rats. Both stress and U69,593-induced allodynia were prevented by right CeA U0126, a mitogen-activated protein kinase inhibitor, presumably acting downstream of KOR. Conclusions Our data reveal a novel lateralized KOR circuit that mediated stress-induced cutaneous allodynia and increased plasma CGRP in an injury-free model of functional cephalic pain with features of migraine and medication overuse headache. Selective, small molecule, orally available, and reversible KOR antagonists are currently in development and may represent a novel class of preventive therapeutics for migraine.

Objective The objective of this study was the determination of the role of calcitonin gene-related peptide (CGRP) in the induction of medication overuse headache (MOH)-related migraine in an injury-free preclinical model. Methods Rats were primed by a 7-day period of exposure to acute migraine therapies including sumatriptan and morphine. After an additional 14-day drug-free period, rats were exposed to putative migraine triggers including bright light stress (BLS) or nitric oxide (NO) donor in the presence or absence of TEV48125, a fully humanized CGRP antibody. Cutaneous allodynia (CA) was used as an outcome measure and CGRP blood and cerebrospinal fluid (CSF) levels were measured. Results BLS and NO donor challenge evoked delayed, long-lasting CA selectively in rats that were previously treated with sumatriptan or morphine. BLS produced a significant increase in CGRP in the plasma, but not CSF, in animals that were previously exposed to sumatriptan compared to saline controls. TEV48125 did not modify baseline tactile thresholds or produce behavioral side effects, but significantly inhibited both BLS- and NO donor-induced CA in animals that were previously primed with sumatriptan or morphine; an isotype control protein that does not bind CGRP had no effect. Interpretation These data suggest that acute migraine medications may promote MOH in susceptible individuals through CGRP-dependent mechanisms and that anti-CGRP antibodies may be a useful clinical strategy for the treatment of MOH.