LY3295668 chemical characterization

Time-dependent increase in apoptosis induction by LY3214996, pan-RAF inhibitor LY3009120 and MEK inhibitor trametinib in KRAS-mutant HCT116 colorectal cancer cells.

Supplementary Figure S1. Validation of BRAF deletion in BxPC-3 cells by Sanger's sequencing method. Supplementary Figure S2. BRAF deletion (deltaBRAF), but not WT BRAF, is able to transform cells. Supplementary Figure S3. Validation of in situ PLA in HeLa and HEK293 cells. Supplementary Figure S4. Phospho-CRAF and MAPK activation in selected tumor cells with BRAF alteration or KRAS mutation. Supplementary Figure S5. Phospho-MEK and ERK inhibition by dabrafenib in tumor cells harboring BRAF deletion. Supplementary Figure S6. In vitro effects of MAPK inhibitors on tumor cells harboring BRAF deletions. Supplementary Table S1. Novel somatic BRAF in-frame deletions from cancer cell lines and patient samples. Supplementary Figure S7. LY3009120, but not vemurafenib, exhibited significant tumor growth inhibition and regression in lung and pancreatic tumor xenograft models harboring the BRAF deletions without significant body weight loss. Supplementary Table S2. IC50 of LY3009120 and vemurafenib against tumor cells with atypical BRAF mutations.

Abs IC50 of LY3214996 and ERK pathway alterations in 60 cancer cell lines profiled for antitumor activity in cell proliferation assay

Biochemical and cellular selectivity profile of LY3214996 (DiscoveRx and ActiveX data)

Figure Legends to supplementary Figures 1-5

Effect of LY3214996 on body weight change and calculation of %dT/C in HCT116 xenograft model.

IL-13 is the primary upregulated cytokine in atopic dermatitis (AD) skin and is the pathogenic mediator driving AD pathophysiology. Lebrikizumab, tralokinumab and cendakimab are therapeutic monoclonal antibodies (mAb) that target IL-13. We undertook studies to compare in vitro binding affinities and cell-based functional activities of lebrikizumab, tralokinumab and cendakimab. Lebrikizumab bound IL-13 with higher affinity (as determined using surface plasma resonance) and slower off-rate. It was more potent in neutralizing IL-13-induced effects in STAT6 reporter and primary dermal fibroblast periostin secretion assays than either tralokinumab or cendakimab. Live imaging confocal microscopy was employed to determine the mAb effects on IL-13 internalization into cells via the decoy receptor IL-13Rα2, using A375 and HaCaT cells. The results showed that only the IL-13/lebrikizumab complex was internalized and co-localized with lysosomes, whereas IL-13/tralokinumab or IL-13/cendakimab complexes did not internalize. Lebrikizumab is a potent, neutralizing high-affinity antibody with a slow disassociation rate from IL-13. Additionally, lebrikizumab does not interfere with IL-13 clearance. Lebrikizumab has a different mode of action to both tralokinumab and cendakimab, possibly contributing to the clinical efficacy observed by lebrikizumab in Ph2b/3 AD studies.

Abs IC50 values (µM) for 36 small molecule inhibitors across a panel of cancer cell lines.

Supplementary figure S1 describes the molecular mechanisms of resistance across vemurafenib-resistant cell line models. Supplementary figure S2 serves as a repeat of figure 3a describing development of in vivo model of resistance, and demonstrates the effect of vemurafenib withdrawl on the resistant tumors. Supplementary figure S3 describes the effect of vemurafenib treatment on MAPK pathway inhibition in A375-RV2 cells. Supplementary figure S4 shows that selective cyclin D1 knockdown or CDK4/6 inhibition induces apoptosis in A375-R1 and M14-R vemurafenib-resistant melanoma cells.

Supplementary Figure 1 (A-C). LY2523355 has potent and selective activity against microtubule-stimulated Eg5 ATPase activity but unlike antitubulins, has no effect on microtubule dynamics in vitro; supplementary Figure 2 (A-E). LY2523355 causes cell cycle arrest at mitosis with monopolar spindle and induces apoptosis; supplementary Figure 3 (A-D). Induction of apoptosis by LY2523355 requires sustained spindle assembly checkpoint (SAC)-mediated mitotic arrest; supplementary Figure 4 (A-D). Antitumor activity of LY2523355 in patient-derived xenograft (PDX) models is correlated with mitotic arrest of cancer cells; supplementary Figure 5 (A,B). Mitotic arrest and apotosis of p388 cells in ascities fluid after continuous infusion of LY2523355, demonstrating the threshold concentration effect.

Generation of vemurafenib-resistant A375 model in vivo.

Abs IC50 values (µM) for LY3295668 across a panel of 580 cell lines.

Supplementary table S1 describes the copy number variations in A375-R1 and A375 parental cells. Supplementary figure legends describe supplementary figures S1-3.

pan-RAF inhibitor can block increase in ERK phosphorylation due to inhibition of negative feedback on CRAF by ERK inhibitor.

In vivo effect of treatment on body weight loss.