The glycocalyx component and sialomucin podocalyxin (PODXL) is required for normal tissue development by promoting apical membranes to form between cells, triggering lumen formation. Elevated PODXL expression is also associated with metastasis and poor clinical outcome in multiple tumor types. How PODXL presents this duality in effect remains unknown. We identify an unexpected function of PODXL as a decoy receptor for galectin-3 (GAL3), whereby the PODXL-GAL3 interaction releases GAL3 repression of integrin-based invasion. Differential cortical targeting of PODXL, regulated by ubiquitination, is the molecular mechanism controlling alternate fates. Both PODXL high and low surface levels occur in parallel subpopulations within cancer cells. Orthotopic intraprostatic xenograft of PODXL-manipulated cells or those with different surface levels of PODXL define that this axis controls metastasis in vivo. Clinically, interplay between PODXL-GAL3 stratifies prostate cancer patients with poor outcome. Our studies define the molecular mechanisms and context in which PODXL promotes invasion and metastasis.

Altered eIF4A1 activity promotes translation of highly structured, eIF4A1-dependent oncogene mRNAs at root of oncogenic translational programmes. It remains unclear how these mRNAs recruit and activate eIF4A1 unwinding specifically to facilitate their preferential translation. Here, we show that single-stranded RNA sequence motifs specifically activate eIF4A1 unwinding allowing local RNA structural rearrangement and translation of eIF4A1-dependent mRNAs in cells. Our data demonstrate that eIF4A1-dependent mRNAs contain AG-rich motifs within their 5'UTR which specifically activate eIF4A1 unwinding of local RNA structure to facilitate translation. This mode of eIF4A1 regulation is used by mRNAs encoding components of mTORC-signalling and cell cycle progression, and renders these mRNAs particularly sensitive to eIF4A1-inhibition. Mechanistically, we show that binding of eIF4A1 to AG-rich sequences leads to multimerization of eIF4A1 with eIF4A1 subunits performing distinct enzymatic activities. Our structural data suggest that RNA-binding of multimeric eIF4A1 induces conformational changes in the RNA resulting in an optimal positioning of eIF4A1 proximal to the RNA duplex enabling efficient unwinding. Our data proposes a model in which AG-motifs in the 5'UTR of eIF4A1-dependent mRNAs specifically activate eIF4A1, enabling assembly of the helicase-competent multimeric eIF4A1 complex, and positioning these complexes proximal to stable localised RNA structure allowing ribosomal subunit scanning.

The mitochondrial genome encodes essential machinery for respiration and metabolic homeostasis but is paradoxically among the most common targets of somatic mutation in the cancer genome, with truncating mutations in respiratory complex I genes being most over-represented 1 . While mitochondrial DNA (mtDNA) mutations have been associated with both improved and worsened prognoses in several tumour lineages 1-,3 , whether these mutations are drivers or exert any functional effect on tumour biology remains controversial. Here we discovered that complex I-encoding mtDNA mutations are sufficient to remodel the tumour immune landscape and therapeutic resistance to immune checkpoint blockade. Using mtDNA base editing technology 4 we engineered recurrent truncating mutations in the mtDNA-encoded complex I gene, Mt-Nd5 , into murine models of melanoma. Mechanistically, these mutations promoted utilisation of pyruvate as a terminal electron acceptor and increased glycolytic flux without major effects on oxygen consumption, driven by an over-reduced NAD pool and NADH shuttling between GAPDH and MDH1, mediating a Warburg-like metabolic shift. In turn, without modifying tumour growth, this altered cancer cell-intrinsic metabolism reshaped the tumour microenvironment in both mice and humans, promoting an anti- tumour immune response characterised by loss of resident neutrophils. This subsequently sensitised tumours bearing high mtDNA mutant heteroplasmy to immune checkpoint blockade, with phenocopy of key metabolic changes being sufficient to mediate this effect. Strikingly, patient lesions bearing >50% mtDNA mutation heteroplasmy also demonstrated a >2.5-fold improved response rate to checkpoint inhibitor blockade. Taken together these data nominate mtDNA mutations as functional regulators of cancer metabolism and tumour biology, with potential for therapeutic exploitation and treatment stratification.

The murine helminth parasite Heligmosomoides polygyrus expresses a family of modular proteins which, replicating the functional activity of the immunomodulatory cytokine TGF-β, have been named TGM (TGF-β Μimic). Multiple domains bind to different receptors, including TGF-β receptors TβRI (ALK5) and TβRII through domains 1-3, and prototypic family member TGM1 binds the cell surface co-receptor CD44 through domains 4-5. This allows TGM1 to induce T lymphocyte Foxp3 expression, characteristic of regulatory (Treg) cells, and to activate a range of TGF-β-responsive cell types. In contrast, a related protein, TGM4, targets a much more restricted cell repertoire, primarily acting on myeloid cells, with less potent effects on T cells and lacking activity on other TGF-β-responsive cell types. TGM4 binds avidly to myeloid cells by flow cytometry, and can outcompete TGM1 for cell binding. Analysis of receptor binding in comparison to TGM1 reveals a 10-fold higher affinity than TGM1 for TGFβR-I (TβRI), but a 100-fold lower affinity for TβRII through Domain 3. Consequently, TGM4 is more dependent on co-receptor binding; in addition to CD44, TGM4 also engages CD49d (Itga4) through Domains 1-3, as well as CD206 and Neuropilin-1 through Domains 4 and 5. TGM4 was found to effectively modulate macrophage populations, inhibiting lipopolysaccharide-driven inflammatory cytokine production and boosting interleukin (IL)-4-stimulated responses such as Arginase-1 in vitro and in vivo. These results reveal that the modular nature of TGMs has allowed the fine tuning of the binding affinities of the TβR- and co-receptor binding domains to establish cell specificity for TGF-β signalling in a manner that cannot be attained by the mammalian cytokine.

Macrophages are fundamental cells of the innate immune system that support normal haematopoiesis and play roles in both anti-cancer immunity and tumour progression. Here we use a chimeric mouse model of chronic myeloid leukaemia (CML) and human bone marrow (BM) derived macrophages to study the impact of the dysregulated BM microenvironment on bystander macrophages. Utilising single-cell RNA sequencing (scRNA-seq) of Philadelphia chromosome (Ph) negative macrophages we reveal unique subpopulations of immature macrophages residing in the CML BM microenvironment. CML exposed macrophages separate from their normal counterparts by reduced expression of the surface marker CD36, which significantly reduces clearance of apoptotic cells. We uncover aberrant production of CML-secreted factors, including the immune modulatory protein lactotransferrin (LTF), that suppresses efferocytosis, phagocytosis, and CD36 surface expression in BM macrophages, indicating that the elevated secretion of LTF is, at least partially responsible for the supressed clearance function of Ph- macrophages.

The mitochondrial genome (mtDNA) encodes essential machinery for oxidative phosphorylation and metabolic homeostasis. Tumor mtDNA is among the most somatically mutated regions of the cancer genome, but whether these mutations impact tumor biology is debated. We engineered truncating mutations of the mtDNA-encoded complex I gene, Mt-Nd5, into several murine models of melanoma. These mutations promoted a Warburg-like metabolic shift that reshaped tumor microenvironments in both mice and humans, consistently eliciting an anti-tumor immune response characterized by loss of resident neutrophils. Tumors bearing mtDNA mutations were sensitized to checkpoint blockade in a neutrophil-dependent manner, with induction of redox imbalance being sufficient to induce this effect in mtDNA wild-type tumors. Patient lesions bearing >50% mtDNA mutation heteroplasmy demonstrated a response rate to checkpoint blockade that was improved by ~2.5-fold over mtDNA wild-type cancer. These data nominate mtDNA mutations as functional regulators of cancer metabolism and tumor biology, with potential for therapeutic exploitation and treatment stratification.