Missense variants in the N-terminal domain of the A isoform of FHF2/FGF13 cause an X-linked developmental and epileptic encephalopathy.

Fibroblast growth factor homologous factors (FHFs) are intracellular proteins which regulate voltage-gated sodium (Nav) channels in the brain and other tissues. FHF dysfunction has been linked to neurological disorders including epilepsy. Here, we describe two sibling pairs and three unrelated males who presented in infancy with intractable focal seizures and severe developmental delay. Whole-exome sequencing identified hemi- and heterozygous variants in the N-terminal domain of the A isoform of FHF2 (FHF2A). The X-linked FHF2 gene (also known as FGF13) has alternative first exons which produce multiple protein isoforms that differ in their N-terminal sequence. The variants were located at highly conserved residues in the FHF2A inactivation particle that competes with the intrinsic fast inactivation mechanism of Nav channels. Functional characterization of mutant FHF2A co-expressed with wild-type Nav1.6 (SCN8A) revealed that mutant FHF2A proteins lost the ability to induce rapid-onset, long-term blockade of the channel while retaining pro-excitatory properties. These gain-of-function effects are likely to increase neuronal excitability consistent with the epileptic potential of FHF2 variants. Our findings demonstrate that FHF2 variants are a cause of infantile-onset developmental and epileptic encephalopathy and underline the critical role of the FHF2A isoform in regulating Nav channel function.

Conditional in vivo deletion of LYN kinase has little effect on a BRCA1 loss-of-function-associated mammary tumour model.

LYN kinase is expressed in BRCA1 loss-of-function-dependent mouse mammary tumours, in the cells of origin of such tumours, and in human breast cancer. Suppressing LYN kinase activity in BRCA1-defective cell lines as well as in in vitro cultures of Brca1-null mouse mammary tumours is deleterious to their growth. Here, we examined the interaction between LYN kinase and BRCA1 loss-of-function in an in vivo mouse mammary tumour model, using conditional knockout Brca1 and Lyn alleles. Comparison of Brca1 tumour cohorts showed little difference in mammary tumour formation between animals that were wild type, heterozygous or homozygous for the conditional Lyn allele, although this was confounded by factors including incomplete Lyn recombination in some tumours. RNA-sequencing analysis demonstrated that tumours with high levels of Lyn gene expression had a slower doubling time, but this was not correlated with levels of LYN staining in tumour cells themselves. Rather, high Lyn expression and slower tumour growth were likely a result of B-cell infiltration. The multifaceted role of LYN indicates that it is likely to present difficulties as a therapeutic target in breast cancer.

Enhanced bacterial cancer therapy delivering therapeutic RNA interference of c-Myc.

BACKGROUND: Bacterial cancer therapy was first trialled in patients at the end of the nineteenth century. More recently, tumour-targeting bacteria have been harnessed to deliver plasmid-expressed therapeutic interfering RNA to a range of solid tumours. A major limitation to clinical translation of this is the short-term nature of RNA interference in vivo due to plasmid instability. To overcome this, we sought to develop tumour-targeting attenuated bacteria that stably express shRNA by virtue of integration of an expression cassette within the bacterial chromosome and demonstrate therapeutic efficacy in vitro and in vivo. RESULTS: The attenuated tumour targeting Salmonella typhimurium SL7207 strain was modified to carry chromosomally integrated shRNA expression cassettes at the xylA locus. The colorectal cancer cell lines SW480, HCT116 and breast cancer cell line MCF7 were used to demonstrate the ability of these modified strains to perform intracellular infection and deliver effective RNA and protein knockdown of the target gene c-Myc. In vivo therapeutic efficacy was demonstrated using the Lgr5creERT2Apcflx/flx and BlgCreBrca2flx/flp53flx/flx orthotopic immunocompetent mouse models of colorectal and breast cancer, respectively. In vitro co-cultures of breast and colorectal cancer cell lines with modified SL7207 demonstrated a significant 50-95% (P < 0.01) reduction in RNA and protein expression with SL7207/c-Myc targeted strains. In vivo, following establishment of tumour tissue, a single intra-peritoneal administration of 1 × 106 CFU of SL7207/c-Myc was sufficient to permit tumour colonisation and significantly extend survival with no overt toxicity in control animals. CONCLUSIONS: In summary we have demonstrated that tumour tropic bacteria can be modified to safely deliver therapeutic levels of gene knockdown. This technology has the potential to specifically target primary and secondary solid tumours with personalised therapeutic payloads, providing new multi-cancer detection and treatment options with minimal off-target effects. Further understanding of the tropism mechanisms and impact on host immunity and microbiome is required to progress to clinical translation.

Modification of Diet to Reduce the Stemness and Tumorigenicity of Murine and Human Intestinal Cells.

SCOPE: Black raspberries (BRBs) have colorectal cancer (CRC) chemo-preventative effects. As CRC originates from an intestinal stem cell (ISC) this study has investigated the impact of BRBs on normal and mutant ISCs. METHODS AND RESULTS: Mice with an inducible Apcfl mutation in either the ISC (Lgr5CreERT2 ) or intestinal crypt (AhCre/VillinCreERT2 ) are fed a control or 10% BRB-supplemented diet. This study uses immunohistochemistry, gene expression analysis, and organoid culture to evaluate the effect of BRBs on intestinal homeostasis. RNAscope is performed for ISC markers on CRC adjacent normal colonic tissue pre and post BRB intervention from patients. 10% BRB diet has no overt effect on murine intestinal homeostasis, despite a reduced stem cell number. Following Apc ISC deletion, BRB diet extends lifespan and reduces tumor area. In the AhCre model, BRB diet attenuates the "crypt-progenitor" phenotype and reduces ISC marker gene expression. In ex vivo culture BRBs reduce the self-renewal capacity of murine and human Apc deficient organoids. Finally, the study observes a reduction in ISC marker gene expression in adjacent normal crypts following introduction of BRBs to the human bowel. CONCLUSION: BRBs play a role in CRC chemoprevention by protectively regulating the ISC compartment and further supports the use of BRBs in CRC prevention.