Abstract B030: Overcoming off-target resistance to therapy in tumors driven by kinase fusions

Abstract Background: Sequential Tyrosine Kinase Inhibitor (TKI) therapy is a well-established strategy to overcome on-target resistance in ALK, ROS1, TRK, and RET kinase fusion-positive (+) tumors. However, this approach does not address off-target resistance that occurs when tumors acquire oncogenic alterations that activate bypass signaling pathways. A hallmark of off-target resistance is the sustained activation of ERK, a process driven by the hyperactivation of Receptor Tyrosine Kinases (RTKs) and/or acquired alterations within the downstream RAS-MAPK signaling pathway. Hypothesis: We hypothesize that inhibition of the MAPK/ERK pathway (using SHP2i or ERKi) can overcome off-target resistance by resensitizing tumors to their original TKIs. Methods: We generated multiple transduced and patient-derived kinase fusion+ cell lines and PDXs with off-target resistance to TKIs. These included ALK, TRK, and RET fusion+ models carrying MET or IGF1R amplification as well as ALK, TRK, and ROS1 fusion+ models carrying a KRAS mutation. The activity and safety of next-generation ERKi (ulixertinib, temuterkib, ERAS-007) or SHP2i (TNO-155, BBP-398, ERAS-601) as monotherapies or combined with TKIs was tested using viability assays, synergy tests, downstream signaling profiling, and in vivo tolerability and efficacy studies. Results: ERK and SHP2 inhibition was able to resensitize all models to the original TKIs in vitro. Among the SHP2is tested, TNO-155 and ERAS-601 were equally active, and both synergized (BLISS score >10) with TKIs (e.g., BLISS scores of 23.38 and 33.88 for TNO+TKI and 601+TKI, respectively in an ALK+, IGF1R amplified model). Among ERKis, only ERAS-007 resensitized these cell lines to TKIs, with BLISS scores up to 25.36. Notably, ERAS-007 also demonstrated potent single-agent activity at nanomolar concentrations in most models (e.g., 90.6% cell growth inhibition at 25 nM in an ALK+, KRAS mutant cell line; P=.000018). Mechanistically, dual inhibition using SHP2i or ERAS-007 in combination with TKIs completely suppressed phosphorylation of RSK1, a direct downstream substrate of ERK. To determine the optimal drugs’ doses for the in vivo testing, we performed a 12-day tolerability study in healthy mice and found that standard doses of TKIs combined with 30mg/kg QD of SHP2i or ERKi did not cause toxicity (no mouse weight loss or impaired liver/kidney function). Importantly, these combinatorial regimens were significantly more effective than each of the single agents in inhibiting tumor growth in xenografts derived from an ALK+, IGF1R amplified NSCLC cell line (P=.006584 for SHP2i+TKI and P=.013847 for ERKi+TKI) and in a PDX derived from a TRK+, KRAS G12D mutant tumor (P