CARMIL1-AA selectively inhibits macropinocytosis while sparing autophagy

Macropinocytosis is reported to fuel tumor growth and drug resistance by allowing cancer cells to scavenge extracellular macromolecules. However, accurately defining the role of macropinocytosis in cancer depends on our ability to selectively block this process. 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) is widely used to inhibit macropinocytosis but affects multiple Na⁺/H⁺ exchangers (NHE) that regulate cytoplasmic and organellar pH. Consistent with this, we report that EIPA slows proliferation to a greater extent than can be accounted for by macropinocytosis inhibition and triggers conjugation of ATG8 to single membranes (CASM). Knocking down only NHE1 would not avoid macropinocytosis-independent effects on pH. Moreover, contrary to published reports, NHE1 loss did not block macropinocytosis in multiple cell lines. Knocking down CARMIL1 with CRISPR-Cas9 editing limited macropinocytosis, but only by 50%. In contrast, expressing the CARMIL1-AA mutant inhibits macropinocytosis induced by a wide range of macropinocytic stimuli to a similar extent as EIPA. CARMIL1-AA expression did not inhibit proliferation, highlighting the shortcomings of EIPA as a macropinocytosis inhibitor. Importantly, autophagy, another actin dependent, nutrient-producing process, was not affected by CARMIL1-AA expression. In sum, constitutive or inducible CARMIL1-AA expression reduced macropinocytosis without affecting proliferation, RAC activation, or autophagy, other processes that drive tumor initiation and progression.