Genome-wide CRISPR/Cas9 Screens Reveal Shared and Bespoke Mechanisms of Resistance to SHP2 inhibition

SHP2 ( PTPN11 ) acts upstream of SOS1/2 to enable RAS activation. Allosteric inhibitors (SHP2is) stabilize SHP2 auto-inhibition, preventing activation by upstream stimuli. SHP2is block proliferation of RTK- or cycling RAS mutant-driven cancers and overcome adaptive resistance to other RAS-ERK pathway drugs. Several SHP2is are in clinical trials. To identify potential SHP2i resistance mechanisms, we performed genome-wide CRISPR/Cas9 knockout screens on two SHP2i-sensitive AML cell lines and recovered genes expected to cause resistance, including tumor suppressor ( NF1 , PTEN , CDKN1B ) and “RASopathy” ( LZTR1 , RASA2 ) genes, and several novel targets ( INPPL1 , MAP4K5, epigenetic modifiers). We then screened 14 cancer lines with a focused CRISPR library targeting common “hits” from the genome-wide screens. LZTR1 deletion conferred resistance in 12/14 lines, followed by MAP4K5 (8/14), SPRED2 (6/14), STK40 (6/14), and INPPL1 (5/14). INPPL1 , MAP4K5 , or LZTR1 deletion reactivated ERK signaling. INPPL1-mediated sensitization to SHP2i required its NPXY motif but not its lipid phosphatase domain. MAP4K5 acted upstream of MEK via a kinase-dependent target(s), whereas LZTR1 showed cell-dependent effects on RIT and RAS stability. INPPLI , MAP4K5 , or LZTR1 deletion also conferred SHP2i resistance in mice. Our results reveal multiple SHP2i resistance genes, emphasizing the need for detailed understanding of the resistance landscape to arrive at effective combinations. ### Competing Interest Statement B.G.N. is a founder of, holds equity in, and receives consulting fees from Navire Pharm and Lighthorse Therapeutics, and is a founder of and holds equity in Northern Biologics, LP. He also receives consulting fees and equity from Arvinas, Inc., and holds equity in Recursion Pharma and received consulting fees from MPM Capital. His spouse holds equity in Amgen, Inc. and held equity in Moderna and Regeneron at times during this study. N.E.S. is an advisor of Vertex and Qiagen and is a co-founder and advisor of OverT Bio.