Predictive modeling of resistance to SMO inhibition in a patient-derived orthotopic xenograft model of SHH medulloblastoma.

Background: Inhibition of the sonic hedgehog (SHH) pathway with Smoothened (SMO) inhibitors is a promising treatment strategy in SHH-activated medulloblastoma, especially in adult patients. However, the problem is that tumors frequently acquire resistance to the treatment. To understand the underlying resistance mechanisms and to find ways to overcome the resistance, preclinical models that became resistant to SMO inhibition are needed. Methods: To induce SMO inhibitor resistant tumors, we have treated a patient-derived xenograft (PDX) model of SHH medulloblastoma, sensitive to SMO inhibition, with 20 mg/kg Sonidegib using an intermitted treatment schedule. Vehicle-treated and resistant models were subjected to whole-genome and RNA sequencing for molecular characterization and target engagement. In vitro drug screens (76 drugs) were performed using Sonidegib-sensitive and -resistant lines to find other drugs to target the resistant lines. One of the top hits was then validated in vivo. Results: Nine independent Sonidegib-resistant PDX lines were generated. Molecular characterization of the resistant models showed that eight models developed missense mutations in SMO and one gained an inactivating point mutation in MEGF8, which acts downstream of SMO as a repressor in the SHH pathway. The in vitro drug screen with Sonidegib-sensitive and -resistant lines identified good efficacy for Selinexor in the resistant line. Indeed, in vivo treatment with Selinexor revealed that it is more effective in resistant than in sensitive models. Conclusions: We report the first human SMO inhibitor resistant medulloblastoma PDX models, which can be used for further preclinical experiments to develop the best strategies to overcome the resistance to SMO inhibitors in patients.

Influence of closed incision negative-pressure therapy on abdominal donor-site morbidity in microsurgical breast reconstruction.

INTRODUCTION: Closed incision negative-pressure therapy (CINPT) has been shown to shorten the time to heal in post-bariatric abdominoplasty and to lower seroma rates in cosmetic abdominoplasty. The objective of this study was to assess the effect of CINPT on donor-site morbidity following abdominal-based free-flap breast reconstruction. PATIENTS AND METHODS: We reviewed medical records from 225 women who had undergone 300 microsurgical free-flap breast reconstructions from the abdomen from November 1, 2007 to March 31, 2019. Patients were grouped according to wound therapy, including 127 patients in the standard of care group and 98 patients in the CINPT group. Primary outcomes were minor (non-operative) and major (operative) surgical site complications. Secondary outcomes were time to drain removal, in-hospital length, and scar quality. RESULTS: Analysis of patient demographics showed an equal distribution with regard to the age, smoking status, prevalence of diabetes mellitus, preoperative chemotherapy, and previous abdominal surgery in both groups. Significantly more patients with obesity (29.6 vs. 15.8%; p = .01) and bilateral breast reconstruction (40.8 vs. 27.6%; p = .04) were included in the CINPT group. Compared to standard of care, the CINPT group had a lower incidence of major surgical site complications (26.0 vs. 11.2%; p = .001). There was no difference in minor surgical site complications and secondary outcomes between groups. CONCLUSION: The CINPT represents a reliable tool to reduce surgical site complications on the abdominal donor-site in abdominal-based free-flap breast reconstruction.

Safety of a Modified Lipoabdominoplasty Technique for Donor-Site Closure in Abdominal-Based Free Flap Breast Reconstruction.

BACKGROUND: The aesthetic and functional outcomes of the donor site following abdominal-based free flap breast reconstruction have been suboptimal. The objective of this study is to evaluate a modified liposuction-assisted abdominoplasty technique combined with rectus plication (LPARSP) adopted from cosmetic abdominoplasty practice. PATIENTS AND METHODS: All abdominal-based free flap breast reconstructions from 01/2017 to 03/2019 were reviewed. Patients with central fullness and sufficient tissue surplus on the abdomen, thighs and flanks who received LPARSP and rectus plication were identified (LPARSP group) and matched for age and body mass index with patients who underwent conventional abdominoplasty (CA group). Abdominal skin sensation, objective functional and aesthetic measures of the abdomen, as well as patient-reported outcomes (Breast-Q), were analyzed. RESULTS: A total of 28 patients were included. Groups were similar in demographics. The mean amount of lipoaspirate in the LPARSP group was 1054±613.5 ml. The postoperative course was similar in both groups. The LPARSP technique resulted in a lower positioned horizontal scar (p = 0.03). The aesthetic outcome was superior in the LPARSP group (p < 0.0001). Furthermore, the LPARSP group presented with a decreased bulging rate (p = 0.05), and secondary refinement procedures were less frequently demanded (p = 0.02). In addition, the abdominal wall sensation of the flanks was improved in the LPARSP group (p = 0.05), whereby patient-reported outcome measures did not differ between groups. CONCLUSIONS: Lipoabdominoplasty with rectus plication represents a safe approach for donor-site closure in selected patients undergoing abdominal-based free flap breast reconstruction. Superior functional and aesthetic results paired with improved abdominal wall sensation are achieved compared to CA. LEVEL OF EVIDENCE IV: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Functional loss of a noncanonical BCOR-PRC1.1 complex accelerates SHH-driven medulloblastoma formation.

Medulloblastoma is a malignant childhood brain tumor arising from the developing cerebellum. In Sonic Hedgehog (SHH) subgroup medulloblastoma, aberrant activation of SHH signaling causes increased proliferation of granule neuron progenitors (GNPs), and predisposes these cells to tumorigenesis. A second, cooperating genetic hit is often required to push these hyperplastic cells to malignancy and confer mutation-specific characteristics associated with oncogenic signaling. Somatic loss-of-function mutations of the transcriptional corepressor BCOR are recurrent and enriched in SHH medulloblastoma. To investigate BCOR as a putative tumor suppressor, we used a genetically engineered mouse model to delete exons 9/10 of Bcor (BcorΔE9-10 ) in GNPs during development. This mutation leads to reduced expression of C-terminally truncated BCOR (BCORΔE9-10). While BcorΔE9-10 alone did not promote tumorigenesis or affect GNP differentiation, BcorΔE9-10 combined with loss of the SHH receptor gene Ptch1 resulted in fully penetrant medulloblastomas. In Ptch1+/- ;BcorΔE9-10 tumors, the growth factor gene Igf2 was aberrantly up-regulated, and ectopic Igf2 overexpression was sufficient to drive tumorigenesis in Ptch1+/- GNPs. BCOR directly regulates Igf2, likely through the PRC1.1 complex; the repressive histone mark H2AK119Ub is decreased at the Igf2 promoter in Ptch1+/- ;BcorΔE9-10 tumors. Overall, our data suggests that BCOR-PRC1.1 disruption leads to Igf2 overexpression, which transforms preneoplastic cells to malignant tumors.

YAP1 subgroup supratentorial ependymoma requires TEAD and nuclear factor I-mediated transcriptional programmes for tumorigenesis.

YAP1 fusion-positive supratentorial ependymomas predominantly occur in infants, but the molecular mechanisms of oncogenesis are unknown. Here we show YAP1-MAMLD1 fusions are sufficient to drive malignant transformation in mice, and the resulting tumors share histo-molecular characteristics of human ependymomas. Nuclear localization of YAP1-MAMLD1 protein is mediated by MAMLD1 and independent of YAP1-Ser127 phosphorylation. Chromatin immunoprecipitation-sequencing analyses of human YAP1-MAMLD1-positive ependymoma reveal enrichment of NFI and TEAD transcription factor binding site motifs in YAP1-bound regulatory elements, suggesting a role for these transcription factors in YAP1-MAMLD1-driven tumorigenesis. Mutation of the TEAD binding site in the YAP1 fusion or repression of NFI targets prevents tumor induction in mice. Together, these results demonstrate that the YAP1-MAMLD1 fusion functions as an oncogenic driver of ependymoma through recruitment of TEADs and NFIs, indicating a rationale for preclinical studies to block the interaction between YAP1 fusions and NFI and TEAD transcription factors.

Aberrant ERBB4-SRC Signaling as a Hallmark of Group 4 Medulloblastoma Revealed by Integrative Phosphoproteomic Profiling.

The current consensus recognizes four main medulloblastoma subgroups (wingless, Sonic hedgehog, group 3 and group 4). While medulloblastoma subgroups have been characterized extensively at the (epi-)genomic and transcriptomic levels, the proteome and phosphoproteome landscape remain to be comprehensively elucidated. Using quantitative (phospho)-proteomics in primary human medulloblastomas, we unravel distinct posttranscriptional regulation leading to highly divergent oncogenic signaling and kinase activity profiles in groups 3 and 4 medulloblastomas. Specifically, proteomic and phosphoproteomic analyses identify aberrant ERBB4-SRC signaling in group 4. Hence, enforced expression of an activated SRC combined with p53 inactivation induces murine tumors that resemble group 4 medulloblastoma. Therefore, our integrative proteogenomics approach unveils an oncogenic pathway and potential therapeutic vulnerability in the most common medulloblastoma subgroup.

Chd7 is indispensable for mammalian brain development through activation of a neuronal differentiation programme.

Mutations in chromatin modifier genes are frequently associated with neurodevelopmental diseases. We herein demonstrate that the chromodomain helicase DNA-binding protein 7 (Chd7), frequently associated with CHARGE syndrome, is indispensable for normal cerebellar development. Genetic inactivation of Chd7 in cerebellar granule neuron progenitors leads to cerebellar hypoplasia in mice, due to the impairment of granule neuron differentiation, induction of apoptosis and abnormal localization of Purkinje cells, which closely recapitulates known clinical features in the cerebella of CHARGE patients. Combinatory molecular analyses reveal that Chd7 is required for the maintenance of open chromatin and thus activation of genes essential for granule neuron differentiation. We further demonstrate that both Chd7 and Top2b are necessary for the transcription of a set of long neuronal genes in cerebellar granule neurons. Altogether, our comprehensive analyses reveal a mechanism with chromatin remodellers governing brain development via controlling a core transcriptional programme for cell-specific differentiation.

Atypical Teratoid/Rhabdoid Tumors Are Comprised of Three Epigenetic Subgroups with Distinct Enhancer Landscapes.

Atypical teratoid/rhabdoid tumor (ATRT) is one of the most common brain tumors in infants. Although the prognosis of ATRT patients is poor, some patients respond favorably to current treatments, suggesting molecular inter-tumor heterogeneity. To investigate this further, we genetically and epigenetically analyzed 192 ATRTs. Three distinct molecular subgroups of ATRTs, associated with differences in demographics, tumor location, and type of SMARCB1 alterations, were identified. Whole-genome DNA and RNA sequencing found no recurrent mutations in addition to SMARCB1 that would explain the differences between subgroups. Whole-genome bisulfite sequencing and H3K27Ac chromatin-immunoprecipitation sequencing of primary tumors, however, revealed clear differences, leading to the identification of subgroup-specific regulatory networks and potential therapeutic targets.

Active medulloblastoma enhancers reveal subgroup-specific cellular origins.

Medulloblastoma is a highly malignant paediatric brain tumour, often inflicting devastating consequences on the developing child. Genomic studies have revealed four distinct molecular subgroups with divergent biology and clinical behaviour. An understanding of the regulatory circuitry governing the transcriptional landscapes of medulloblastoma subgroups, and how this relates to their respective developmental origins, is lacking. Here, using H3K27ac and BRD4 chromatin immunoprecipitation followed by sequencing (ChIP-seq) coupled with tissue-matched DNA methylation and transcriptome data, we describe the active cis-regulatory landscape across 28 primary medulloblastoma specimens. Analysis of differentially regulated enhancers and super-enhancers reinforced inter-subgroup heterogeneity and revealed novel, clinically relevant insights into medulloblastoma biology. Computational reconstruction of core regulatory circuitry identified a master set of transcription factors, validated by ChIP-seq, that is responsible for subgroup divergence, and implicates candidate cells of origin for Group 4. Our integrated analysis of enhancer elements in a large series of primary tumour samples reveals insights into cis-regulatory architecture, unrecognized dependencies, and cellular origins.

Molecular Classification of Ependymal Tumors across All CNS Compartments, Histopathological Grades, and Age Groups.

Ependymal tumors across age groups are currently classified and graded solely by histopathology. It is, however, commonly accepted that this classification scheme has limited clinical utility based on its lack of reproducibility in predicting patients' outcome. We aimed at establishing a uniform molecular classification using DNA methylation profiling. Nine molecular subgroups were identified in a large cohort of 500 tumors, 3 in each anatomical compartment of the CNS, spine, posterior fossa, supratentorial. Two supratentorial subgroups are characterized by prototypic fusion genes involving RELA and YAP1, respectively. Regarding clinical associations, the molecular classification proposed herein outperforms the current histopathological classification and thus might serve as a basis for the next World Health Organization classification of CNS tumors.

Enhancer hijacking activates GFI1 family oncogenes in medulloblastoma.

Medulloblastoma is a highly malignant paediatric brain tumour currently treated with a combination of surgery, radiation and chemotherapy, posing a considerable burden of toxicity to the developing child. Genomics has illuminated the extensive intertumoral heterogeneity of medulloblastoma, identifying four distinct molecular subgroups. Group 3 and group 4 subgroup medulloblastomas account for most paediatric cases; yet, oncogenic drivers for these subtypes remain largely unidentified. Here we describe a series of prevalent, highly disparate genomic structural variants, restricted to groups 3 and 4, resulting in specific and mutually exclusive activation of the growth factor independent 1 family proto-oncogenes, GFI1 and GFI1B. Somatic structural variants juxtapose GFI1 or GFI1B coding sequences proximal to active enhancer elements, including super-enhancers, instigating oncogenic activity. Our results, supported by evidence from mouse models, identify GFI1 and GFI1B as prominent medulloblastoma oncogenes and implicate 'enhancer hijacking' as an efficient mechanism driving oncogene activation in a childhood cancer.

Genome sequencing of SHH medulloblastoma predicts genotype-related response to smoothened inhibition.

Smoothened (SMO) inhibitors recently entered clinical trials for sonic-hedgehog-driven medulloblastoma (SHH-MB). Clinical response is highly variable. To understand the mechanism(s) of primary resistance and identify pathways cooperating with aberrant SHH signaling, we sequenced and profiled a large cohort of SHH-MBs (n = 133). SHH pathway mutations involved PTCH1 (across all age groups), SUFU (infants, including germline), and SMO (adults). Children >3 years old harbored an excess of downstream MYCN and GLI2 amplifications and frequent TP53 mutations, often in the germline, all of which were rare in infants and adults. Functional assays in different SHH-MB xenograft models demonstrated that SHH-MBs harboring a PTCH1 mutation were responsive to SMO inhibition, whereas tumors harboring an SUFU mutation or MYCN amplification were primarily resistant.

Dissecting the genomic complexity underlying medulloblastoma.

Medulloblastoma is an aggressively growing tumour, arising in the cerebellum or medulla/brain stem. It is the most common malignant brain tumour in children, and shows tremendous biological and clinical heterogeneity. Despite recent treatment advances, approximately 40% of children experience tumour recurrence, and 30% will die from their disease. Those who survive often have a significantly reduced quality of life. Four tumour subgroups with distinct clinical, biological and genetic profiles are currently identified. WNT tumours, showing activated wingless pathway signalling, carry a favourable prognosis under current treatment regimens. SHH tumours show hedgehog pathway activation, and have an intermediate prognosis. Group 3 and 4 tumours are molecularly less well characterized, and also present the greatest clinical challenges. The full repertoire of genetic events driving this distinction, however, remains unclear. Here we describe an integrative deep-sequencing analysis of 125 tumour-normal pairs, conducted as part of the International Cancer Genome Consortium (ICGC) PedBrain Tumor Project. Tetraploidy was identified as a frequent early event in Group 3 and 4 tumours, and a positive correlation between patient age and mutation rate was observed. Several recurrent mutations were identified, both in known medulloblastoma-related genes (CTNNB1, PTCH1, MLL2, SMARCA4) and in genes not previously linked to this tumour (DDX3X, CTDNEP1, KDM6A, TBR1), often in subgroup-specific patterns. RNA sequencing confirmed these alterations, and revealed the expression of what are, to our knowledge, the first medulloblastoma fusion genes identified. Chromatin modifiers were frequently altered across all subgroups. These findings enhance our understanding of the genomic complexity and heterogeneity underlying medulloblastoma, and provide several potential targets for new therapeutics, especially for Group 3 and 4 patients.