Joan K. Heath

Transitioning a candidate therapeutic target from bench to bedside requires significant time and financial investment, yet clinical success remains low often due to poor on-target toxicity assessment during preclinical validation. Tag-degraders provide a tool to improve target validation by enabling degradation of any protein of interest via a degron-tag. This drug-based, reversible, and dose-dependent method of protein removal can mimic degrader-based drug treatments and assess the implications of target protein depletion in vivo. However, each degrader has a distinct pharmacokinetic profile that will influence its effectiveness across tissues. To create a resource to enable the most appropriate choice of tag-degrader, we benchmark the dTAG, HaloPROTAC, and NanoTAC systems in vivo by employing a transgenic mouse expressing a reporter protein targetable by these tag-degraders. Through various treatment regimes, we characterise each degrader profile across a panel of 20 tissues and organs, highlighting the superior degradation by dTAG molecules, and identify differences between degradation in whole tissues versus single cell populations. Using an FKBP^F36V knock-in mouse expressing 65K-FKBP^F36V, we reveal target specific degradation kinetics, and a critical requirement for 65K in mice. Together, this resource will assist researchers in choosing the right degrader and tag for their own applications.

Minor splicing is an under-appreciated splicing system required for the correct expression of ~700 genes in the human genome. This small subset of genes (0.35%) harbours introns containing non-canonical splicing sequences that are recognised uniquely by the minor spliceosome and cannot be processed by the major spliceosome. Using in vivo zebrafish and mouse cancer models, we show that heterozygous expression of Rnpc3 , encoding a unique protein component of the minor spliceosome, restricts the growth and survival of liver, lung and gastric tumours without impacting healthy cells. RNPC3 knockdown in human lung cancer-derived A549 cells also impairs cell proliferation and RNA-seq analysis reveals a robust and selective disruption to minor intron splicing and transcription-wide effects on gene expression. We further demonstrate that these perturbations are accompanied by DNA replication stress, DNA damage, accumulation of TP53 protein and activation of a Tp53-dependent transcriptional program that induces cell cycle arrest and apoptosis. Together our data reveal a vulnerability of cancer cells to minor splicing inhibition that restricts tumour growth.

The nucleoporin (NUP) ELYS, encoded by AHCTF1, is a large multifunctional protein with essential roles in nuclear pore assembly and mitosis. Using both larval and adult zebrafish models of hepatocellular carcinoma (HCC), in which the expression of an inducible mutant kras transgene (kras^G12V) drives hepatocyte-specific hyperplasia and liver enlargement, we show that reducing ahctf1 gene dosage by 50% markedly decreases liver volume, while non-hyperplastic tissues are unaffected. We demonstrate that in the context of cancer, ahctf1 heterozygosity impairs nuclear pore formation, mitotic spindle assembly, and chromosome segregation, leading to DNA damage and activation of a Tp53-dependent transcriptional programme that induces cell death and cell cycle arrest. Heterozygous expression of both ahctf1 and ranbp2 (encoding a second nucleoporin), or treatment of heterozygous ahctf1 larvae with the nucleocytoplasmic transport inhibitor, Selinexor, completely blocks kras^G12V-driven hepatocyte hyperplasia. Gene expression analysis of patient samples in the liver hepatocellular carcinoma (LIHC) dataset in The Cancer Genome Atlas shows that high expression of one or more of the transcripts encoding the 10 components of the NUP107–160 subcomplex, which includes AHCTF1, is positively correlated with worse overall survival. These results provide a strong and feasible rationale for the development of novel cancer therapeutics that target ELYS function and suggest potential avenues for effective combinatorial treatments.

Complex organ development depends on single lumen formation and its expansion during tubulogenesis. This can be achieved by correct mitotic spindle orientation during cell division, combined with luminal fluid filling that generates hydrostatic pressure. Using a human 3D cell culture model, we have identified two regulators of these processes. We find that pleckstrin homology leucine-rich repeat protein phosphatase (PHLPP) 2 regulates mitotic spindle orientation, and thereby midbody positioning and maintenance of a single lumen. Silencing the sole PHLPP family phosphatase in Drosophila melanogaster, phlpp, resulted in defective spindle orientation in Drosophila neuroblasts. Importantly, cystic fibrosis transmembrane conductance regulator (CFTR) is the main channel regulating fluid transport in this system, stimulated by phosphorylation by protein kinase A and inhibited by the AMP-activated protein kinase AMPK. During lumen expansion, CFTR remains open through the action of PHLPP1, which stops activated AMPK from inhibiting ion transport through CFTR. In the absence of PHLPP1, the restraint on AMPK activity is lost and this tips the balance in the favour of channel closing, resulting in the lack of lumen expansion and accumulation of mucus.

Prompted by the occasion of International Women's Day, Joan Heath and DMM reunited Professors Suzanne Cory and Joan Steitz via Zoom to discuss their extraordinary careers and joint experiences in science. They also delve into past and present challenges for women in science, and discuss the role of scientists in a post-pandemic world.

Modulation of protein abundance using tag-Targeted Protein Degrader (tTPD) systems targeting FKBP12^F36V (dTAGs) or HaloTag7 (HaloPROTACs) are powerful approaches for preclinical target validation. Interchanging tags and tag-targeting degraders is important to achieve efficient substrate degradation, yet limited degrader/tag pairs are available and side-by-side comparisons have not been performed. To expand the tTPD repertoire we developed catalytic NanoLuc-targeting PROTACs (NanoTACs) to hijack the CRL4^CRBN complex and degrade NanoLuc tagged substrates, enabling rapid luminescence-based degradation screening. To benchmark NanoTACs against existing tTPD systems we use an interchangeable reporter system to comparatively test optimal degrader/tag pairs. Overall, we find the dTAG system exhibits superior degradation. To align tag-induced degradation with physiology we demonstrate that NanoTACs limit MLKL-driven necroptosis. In this work we extend the tTPD platform to include NanoTACs adding flexibility to tTPD studies, and benchmark each tTPD system to highlight the importance of comparing each system against each substrate.

Minor splicing is a second splicing system required for the correct expression of ∼700 human minor intron-containing genes (MIGs). Many MIGs are expressed in vigorously proliferating cells and are frequently dysregulated in cancer including BRAF, ERK, JNK and p38 . Minor splicing is carried out by the minor spliceosome which comprises several unique components, including a 65kDa protein encoded by RNPC3 . We show that Rnpc3 heterozygosity reduces tumour burden in a broad spectrum of in vivo cancer settings, without harming normal tissues. Using the collective power of zebrafish, mouse and human cancer models, we reveal a sequence of events connecting Rnpc3 deficiency and impaired splicing of MIGs to DNA damage and activation of a Tp53-dependent transcriptional program that restricts tumour burden by inducing cell cycle arrest and apoptosis. Interrogation of human liver and lung cancer transcriptomes curated in TCGA revealed that the expression of many of the genes encoding protein components of the minor spliceosome is upregulated in these cancers. This is accompanied by upregulation of the expression of MIGs that are enriched in cell cycle and DNA damage pathways. These findings suggest that cancer cells can invoke mechanisms to increase the efficiency of minor splicing to support their high proliferation rates. Finally, Kaplan Meier survival analysis shows that highly expressed MIGs are frequently associated with poor patient survival. Taken together, these results indicate that the minor spliceosome offers a therapeutically viable target for the treatment of a broad spectrum of cancers.

The development of a functional vasculature requires the coordinated control of cell fate, lineage differentiation and network growth. Cellular proliferation is spatiotemporally regulated in developing vessels, but how this is orchestrated in different lineages is unknown. Here, using a zebrafish genetic screen for lymphatic-deficient mutants, we uncover a mutant for the RNA helicase Ddx21. Ddx21 cell-autonomously regulates lymphatic vessel development. An established regulator of ribosomal RNA synthesis and ribosome biogenesis, Ddx21 is enriched in sprouting venous endothelial cells in response to Vegfc–Flt4 signalling. Ddx21 function is essential for Vegfc–Flt4-driven endothelial cell proliferation. In the absence of Ddx21, endothelial cells show reduced ribosome biogenesis, p53 and p21 upregulation and cell cycle arrest that blocks lymphangiogenesis. Thus, Ddx21 coordinates the lymphatic endothelial cell response to Vegfc–Flt4 signalling by balancing ribosome biogenesis and p53 function. This mechanism may be targetable in diseases of excessive lymphangiogenesis such as cancer metastasis or lymphatic malformation.

The nucleoporin ELYS, encoded by AHCTF1 , is a large multifunctional protein with essential roles in nuclear pore assembly and mitosis. Using a zebrafish model of hepatocellular carcinoma, in which the expression of an inducible mutant kras transgene (kras ^G12V ) drives hepatocyte-specific hyperplasia and liver enlargement, we show that reducing ahctf1 gene dosage by 50% markedly shrinks tumour burden, while non-hyperplastic tissues are unaffected. We demonstrate that ahctf1 heterozygosity impairs nuclear pore formation, mitotic spindle assembly and chromosome segregation, leading to DNA damage and activation of TP53-dependent and independent mechanisms of cell death and cell cycle arrest. This selective vulnerability of cancer cells to mild disruption of Elys function uncovers a novel synthetic lethal interaction between ahctf1 and kras mutations that could be exploited therapeutically. Heterozygous expression of both ahctf1 and ranbp2 , or treatment of heterozygous ahctf1 larvae with the nucleocytoplasmic transport inhibitor, Selinexor, completely blocked kras ^G12V -driven hepatocyte hyperplasia, revealing promising avenues for combinatorial treatments.

Splicing is an essential step in eukaryotic gene expression. While the majority of introns is excised by the U2-dependent, or major class, spliceosome, the appropriate expression of a very small subset of genes depends on U12-dependent, or minor class, splicing. The U11/U12 65K protein (hereafter 65K), encoded by RNPC3, is one of seven proteins that are unique to the U12-dependent spliceosome, and previous studies including our own have established that it plays a role in plant and vertebrate development. To pinpoint the impact of 65K loss during mammalian development and in adulthood, we generated germline and conditional Rnpc3-deficient mice. Homozygous Rnpc3^−/− embryos died prior to blastocyst implantation, whereas Rnpc3^+/− mice were born at the expected frequency, achieved sexual maturity, and exhibited a completely normal lifespan. Systemic recombination of conditional Rnpc3 alleles in adult (Rnpc3^lox/lox) mice caused rapid weight loss, leukopenia, and degeneration of the epithelial lining of the entire gastrointestinal tract, the latter due to increased cell death and a reduction in cell proliferation. Accompanying this, we observed a loss of both 65K and the pro-proliferative phospho-ERK1/2 proteins from the stem/progenitor cells at the base of intestinal crypts. RT-PCR analysis of RNA extracted from purified preparations of intestinal epithelial cells with recombined Rnpc3^lox alleles revealed increased frequency of U12-type intron retention in all transcripts tested. Our study, using a novel conditional mouse model of Rnpc3 deficiency, establishes that U12-dependent splicing is not only important during development but is indispensable throughout life.