Biallelic variants in FLII cause pediatric cardiomyopathy by disrupting cardiomyocyte cell adhesion and myofibril organization

Pediatric cardiomyopathy (CM) represents a group of rare, severe disorders that affect the myocardium. To date, the etiology and mechanisms underlying pediatric CM are incompletely understood, hampering accurate diagnosis and individualized therapy development. Here, we identified biallelic variants in the highly conserved flightless-I (FLII) gene in 3 families with idiopathic, early-onset dilated CM. We demonstrated that patient-specific FLII variants, when brought into the zebrafish genome using CRISPR/Cas9 genome editing, resulted in the manifestation of key aspects of morphological and functional abnormalities of the heart, as observed in our patients. Importantly, using these genetic animal models, complemented with in-depth loss-of-function studies, we provided insights into the function of Flii during ventricular chamber morphogenesis in vivo, including myofibril organization and cardiomyocyte cell adhesion, as well as trabeculation. In addition, we identified Flii function to be important for the regulation of Notch and Hippo signaling, crucial pathways associated with cardiac morphogenesis and function. Taken together, our data provide experimental evidence for a role for FLII in the pathogenesis of pediatric CM and report biallelic variants as a genetic cause of pediatric CM.</p

Combinatorial immunotherapies overcome MYC-driven immune evasion in triple negative breast cancer

Few patients with triple negative breast cancer (TNBC) benefit from immune checkpoint inhibitors with complete and durable remissions being quite rare. Oncogenes can regulate tumor immune infiltration, however whether oncogenes dictate diminished response to immunotherapy and whether these effects are reversible remains poorly understood. Here, we report that TNBCs with elevated MYC expression are resistant to immune checkpoint inhibitor therapy. Using mouse models and patient data, we show that MYC signaling is associated with low tumor cell PD-L1, low overall immune cell infiltration, and low tumor cell MHC-I expression. Restoring interferon signaling in the tumor increases MHC-I expression. By combining a TLR9 agonist and an agonistic antibody against OX40 with anti-PD-L1, mice experience tumor regression and are protected from new TNBC tumor outgrowth. Our findings demonstrate that MYC-dependent immune evasion is reversible and druggable, and when strategically targeted, may improve outcomes for patients treated with immune checkpoint inhibitors. The oncoprotein c-Myc is often overexpressed in triple negative breast cancer and has a role in tumor progression and resistance to therapy. Here the authors show that elevated MYC expression is correlated with low immune infiltration, diminished MHC-I pathway expression and that CpG/aOX40 treatment could overcome resistance to PD-L1 blockade in MYC-high breast tumors.Peer reviewe

Cavin4b/Murcb Is Required for Skeletal Muscle Development and Function in Zebrafish

Skeletal muscles provide metazoans with the ability to feed, reproduce and avoid predators. In humans, a heterogeneous group of genetic diseases, termed muscular dystrophies ( MD), lead to skeletal muscle dysfunction. Mutations in the gene encoding Caveolin-3, a principal component of the membrane micro-domains known as caveolae, cause defects in muscle maintenance and function; however it remains unclear how caveolae dysfunction underlies MD pathology. The Cavin family of caveolar proteins can form membrane remodeling oligomers and thus may also impact skeletal muscle function. Changes in the distribution and function of Cavin4/Murc, which is predominantly expressed in striated muscles, have been reported to alter caveolae structure through interaction with Caveolin-3. Here, we report the generation and phenotypic analysis of murcb mutant zebrafish, which display impaired swimming capacity, skeletal muscle fibrosis and T-tubule abnormalities during development. To understand the mechanistic importance of Murc loss of function, we assessed Caveolin-1 and 3 localization and found it to be abnormal. We further identified an in vivo function for Murc in Erk signaling. These data link Murc with developmental defects in T-tubule formation and progressive muscle dysfunction, thereby providing a new candidate for the etiology of muscular dystrophy

Ultra-structural analysis of Cavin4b/Murcb deficient skeletal muscle.

<p>A. Representative electron micrographs of <i>murcb</i>^{<i>s983/+</i>} and <i>murcb</i>^{<i>s983/s983</i>} zebrafish at 80 hpf. T-tubule triad structures are indicated with a box and enlargements of this region are shown on the left. B. Representative electron micrographs of <i>murcb</i>^{<i>s983/+</i>} and <i>murcb</i>^{<i>s983/s983</i>} zebrafish at 80 hpf. Caveloae are indicated by arrows. Enlargements from the micrographs are shown on the right.</p

Filamentous actin analysis of Cavin4b/Murcb deficient zebrafish.

<p>Representative lateral views of confocal projections of phalloidin stained <i>murcb</i>^{<i>s983/+</i>} and <i>murcb</i>^{<i>s983/s983</i>} zebrafish at 3 dpf and 6 dpf. Skeletal muscle fibers are smaller and less consistent in size in <i>murcb</i> mutant animals compared to wild-type siblings.</p

Swimming analysis of Cavin4b/Murcb deficient zebrafish.

<p>Dot plots of maximum swimming velocity and acceleration following the startle response of 60 hpf, 80 hpf, and 10 wpf <i>murcb</i>^{<i>s983/+</i>} and <i>murcb</i>^{<i>s983/s983</i>} zebrafish. The red line represents the mean. *p<0.05 ***p<0.0005</p

Characterization of Cavin4b/Murcb deficient zebrafish.

<p>A. Representative lateral views of <i>murcb</i>^{<i>s983/s983</i>} and <i>murcb</i>^{<i>s983/+</i>} sibling zebrafish at 10 wpf. Fish in the top panels are females while those in the bottom panels are males. B. Dot plots of body mass and length of 10 wpf wt, <i>murcb</i>^{<i>s983/+</i>}, and <i>murcb</i>^{<i>s983/s983</i>} zebrafish. Length measurements were made from the anterior point to the caudal fin/trunk boundary. Approximately equal numbers of male and female fish were used. The red line represents the mean. ***p<0.0005 C. H&E stain of 10 μm sections prepared from the trunk of 10 wpf <i>murcb</i>^{<i>s983/+</i>} and <i>murcb</i>^{<i>s983/s983</i>} zebrafish. Two examples of <i>murcb</i>^{<i>s983/s983</i>} fish of different sizes are shown. D. Phalloidin staining of 10 μm transverse sections prepared from the trunk of 10 wpf <i>murcb</i>^{<i>s983/+</i>} and <i>murcb</i>^{<i>s983/s983</i>} zebrafish. E. Trichrome staining of 10 μm transverse sections prepared from the trunk of 12 mpf <i>murcb</i>^{<i>s983/+</i>} and <i>murcb</i>^{<i>s983/s983</i>} zebrafish. F. Representative lateral views of <i>murcb</i>^{<i>s983/+</i>} and <i>murcb</i>^{<i>s983/s983</i>} zebrafish at 5 dpf.</p

Transcriptional profiling of Cavin4b/Murcb deficient zebrafish.

<p>Inset: PANTHER Database protein class statistical overrepresentation test on microarray mRNA expression profiling from <i>murcb</i>^{<i>s983/s983</i>} compared to <i>murcb</i>^{<i>s983/+</i>} 72 hpf larvae. 1309 genes with greater than 1.5 fold or less than 0.7 fold expression difference in Cavin4b/Murcb deficient larvae compared to their heterozygous siblings were recognized in the database with 25708 genes comprising the <i>Danio rerio</i> reference list. Protein classes shown had a <i>p</i> value <0.05. The log₂ ratio of <i>murcb</i>^{<i>s983/s983</i>}/<i>murcb</i>^{<i>s983/+</i>} normalized microarray values from genes in the voltage-gated ion channel PANTHER protein class show that 26 members of the voltage-gated ion channel class were misregulated where 10 would be expected. Error bars represent SEM.</p

TALEN design and generation of a <i>murcb</i> mutant allele.

<p>A. RT-PCR analysis of mRNA expression of <i>murca and murcb</i> during zebrafish development. <i>actb1</i> is shown as a loading control. B. RT-PCR analysis of mRNA expression of <i>murca</i> and <i>murcb</i> in adult zebrafish tissues. <i>myl1</i> and <i>myog</i> are shown as controls for skeletal muscle contamination of other tissue samples. <i>actb1</i> is shown as a loading control. C. <i>Whole mount in situ</i> hybridization of <i>murcb</i> mRNA in zebrafish embryos at 48 hpf. Top: lateral view. Bottom left: magnified lateral view. Bottom right: magnified dorsal view. D. TALEN construct for <i>murcb</i> mutagenesis. Sequencing results for the <i>murcb</i>^<i>s983</i> allele. Schematic of wild-type and predicted mutant proteins. The <i>murcb</i>^<i>s983</i> lesion leads to a premature stop codon after 83 missense amino acids starting at amino acid 28. Helical region (HR) domains are indicated in blue. The red arrowhead points to the TALEN target site. The red bar indicates the region of the mutant protein that is out of frame. The green bar indicates the antigen used to generate the antibody used in this work. E. Anti-Cavin4/Murc immunofluorescence (IF) micrographs of 10 μm transverse sections of skeletal muscle prepared from 10 wpf sibling and <i>murcb</i>^{<i>s983/s983</i>} fish. No 1° ab indicates control samples incubated only with secondary antibodies.</p

Combinatorial immunotherapies overcome MYC-driven immune evasion in triple negative breast cancer.

Few patients with triple negative breast cancer (TNBC) benefit from immune checkpoint inhibitors with complete and durable remissions being quite rare. Oncogenes can regulate tumor immune infiltration, however whether oncogenes dictate diminished response to immunotherapy and whether these effects are reversible remains poorly understood. Here, we report that TNBCs with elevated MYC expression are resistant to immune checkpoint inhibitor therapy. Using mouse models and patient data, we show that MYC signaling is associated with low tumor cell PD-L1, low overall immune cell infiltration, and low tumor cell MHC-I expression. Restoring interferon signaling in the tumor increases MHC-I expression. By combining a TLR9 agonist and an agonistic antibody against OX40 with anti-PD-L1, mice experience tumor regression and are protected from new TNBC tumor outgrowth. Our findings demonstrate that MYC-dependent immune evasion is reversible and druggable, and when strategically targeted, may improve outcomes for patients treated with immune checkpoint inhibitors