Rab35 restrains regional Wnt activation by regulating a Cdc42-JNK signalling relay in the intestine. APC-Rab35

Inactivating mutations to Adenomatous Polyposis Coli (Apc) results in uncontrolled Wnt signalling and underlies approx. 80% of colorectal cancer (CRC) cases, yet our understanding of the subcellular events that sustain pathway activity is limited. Here, using a conditional in vivo modifier screen in fly intestinal progenitor cells, we identify Rab35 GTPase as a novel tumour suppressor that modulates regional Wnt signalling after loss of Apc. Single cell RNA sequencing of the intestine when Apc is depleted revealed progenitor-specific increase in a GTPase activating protein, which we named blackbelt, that is important for Wnt activation. Our genetic and subcellular experiments demonstrate that Rab35 surpassingly does not modulate Wnt signalling through influencing Wnt secretion but rather controls the activity and localisation of the Rho GTPase, Cdc42. We identify that the Rab35-Cdc42 axis regulates JNK signalling in order to tune the Wnt pathway upstream of β-Catenin. Importantly, we show that maintaining active JNK signalling is important for the proliferation of Apc mutant mouse colon organoids. Our findings therefore highlight a GTPase cascade that sustains aberrant Wnt activity and provides impetus to exploit this pathway to therapeutically target CRC.