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Supplementary Methods include Enzyme Kinetic Assays and Computational Methods.

Supplementary Figures include Figure S1-S9.

Supplementary Tables 1-2

Supplementary Figures

Supplementary methods, figure legends

Purpose:Current standard initial therapy for advanced, ROS proto-oncogene 1, receptor tyrosine kinase fusion (ROS1)-positive (ROS1+) non–small cell lung cancer (NSCLC) is crizotinib or entrectinib. Lorlatinib, a next-generation anaplastic lymphoma kinase/ROS1 inhibitor, recently demonstrated efficacy in ROS1+ NSCLC, including in crizotinib-pretreated patients. However, mechanisms of lorlatinib resistance in ROS1+ disease remain poorly understood. Here, we assessed mechanisms of resistance to crizotinib and lorlatinib.Experimental Design:Biopsies from patients with ROS1+ NSCLC progressing on crizotinib or lorlatinib were profiled by genetic sequencing.Results:From 55 patients, 47 post-crizotinib and 32 post-lorlatinib biopsies were assessed. Among 42 post-crizotinib and 28 post-lorlatinib biopsies analyzed at distinct timepoints, ROS1 mutations were identified in 38% and 46%, respectively. ROS1 G2032R was the most commonly occurring mutation in approximately one third of cases. Additional ROS1 mutations included D2033N (2.4%) and S1986F (2.4%) post-crizotinib and L2086F (3.6%), G2032R/L2086F (3.6%), G2032R/S1986F/L2086F (3.6%), and S1986F/L2000V (3.6%) post-lorlatinib. Structural modeling predicted ROS1L2086F causes steric interference to lorlatinib, crizotinib, and entrectinib, while it may accommodate cabozantinib. In Ba/F3 models, ROS1L2086F, ROS1G2032R/L2086F, and ROS1S1986F/G2032R/L2086F were refractory to lorlatinib but sensitive to cabozantinib. A patient with disease progression on crizotinib and lorlatinib and ROS1 L2086F received cabozantinib for nearly 11 months with disease control. Among lorlatinib-resistant biopsies, we also identified MET amplification (4%), KRAS G12C (4%), KRAS amplification (4%), NRAS mutation (4%), and MAP2K1 mutation (4%).Conclusions:ROS1 mutations mediate resistance to crizotinib and lorlatinib in more than one third of cases, underscoring the importance of developing next-generation ROS1 inhibitors with potency against these mutations, including G2032R and L2086F. Continued efforts are needed to elucidate ROS1-independent resistance mechanisms.

Lung cancer is one of the most aggressive tumors. Targeted therapies stratified by oncogenic drivers has remarkably improved therapeutic outcomes in patients with non-small cell lung cancer (NSCLC) (1). However, such oncogenic drivers are not found in 25-40% of NSCLC cases (2). Here, we identified a novel fusion transcript of CLIP1 and LTK using whole transcriptome sequencing in a multi-institutional genome screening platform (LC-SCRUM-Asia, UMIN000036871). The CLIP1-LTK fusion was present in 0.4% of NSCLCs and was mutually exclusive with other known oncogenic drivers. We show that kinase activity of CLIP1-LTK fusion protein is constitutively activated and has transformation potential. Treatment of Ba/F3 cells expressing CLIP1-LTK with lorlatinib, an ALK inhibitor, inhibited CLIP1-LTK kinase activity, suppressed proliferation, and induced apoptosis. One NSCLC patient harboring the CLIP1-LTK fusion showed dramatic clinical responses to lorlatinib treatment. To our knowledge, this is the first description of LTK alterations with oncogenic activity in cancers. Our results demonstrate that the CLIP1-LTK fusion is a novel target in NSCLC, which could be treated with lorlatinib.

Lorlatinib is currently the most advanced, potent and selective anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitor for the treatment of ALK-positive non-small cell lung cancer in the clinic; however, diverse compound ALK mutations driving therapy resistance emerge. Here, we determine the spectrum of lorlatinib-resistant compound ALK mutations in patients, following treatment with lorlatinib, the majority of which involve ALK G1202R or I1171N/S/T. We further identify structurally diverse lorlatinib analogs that harbor differential selective profiles against G1202R versus I1171N/S/T compound ALK mutations. Structural analysis revealed increased potency against compound mutations through improved inhibition of either G1202R or I1171N/S/T mutant kinases. Overall, we propose a classification of heterogenous ALK compound mutations enabling the development of distinct therapeutic strategies for precision targeting following sequential tyrosine kinase inhibitors.

The novel pentacyclic polyhydroxylated sterol, xestobergsterol A 1a , a strong inhibitor of histamine release from rat mast cells induced by anti-IgE, has been synthesized in 24 steps and good overall yield from stigmasterol 7 . The Breslow remote functionalization process has been extended to several more highly functionalized steroid derivatives, especially those with oxygen functionality in the B-ring. The key steps of the synthesis of xestobergsterol A 1a and its analogues, 7-deoxyxestobergsterol A 1d and 16,23- seco -23-deoxyxestobergsterol A 73 , are the Breslow remote functionalization of oxygenated steroids and for compounds 1a and 1d , a novel base-catalyzed epimerization-aldol condensation of a dione to give the desired CD- cis ring structure of the xestobergsterols. Thus the known alcohol 75 , prepared from stigmasterol 7 , was taken to the tetraacetate 107 which was then converted via a Breslow remote functionalization into the alkene aldehyde 114 which was transformed in 5 steps to xestobergsterol A 1a . Testing of the synthetic materials showed that the two analogues, 7-deoxyxestobergsterol A 1b and 16,23- seco -23-deoxyxestobergsterol A 73 , are also potent inhibitors of histamine release with IC 50 values (IC 50 500 nM and 750 nM, respectively) being only 1015 times less than that of xestobergsterol A itself (50 nM).

A novel pentacyclic polyhydroxylated sterol, xestobergsterol A (1a), has been synthesized in 24 steps and in good overall yield from stigmasterol 17. The key steps of the synthesis are the Breslow remote functionalization of the polyoxygenated steroid derived from 25 and the base-catalyzed epimerization−aldol condensation of the dione derived from 27.

The novel pentacyclic polyhydroxylated sterol, xestobergsterol A 1a , a strong inhibitor of histamine release from rat mast cells induced by anti-IgE, has been synthesized in 24 steps and good overall yield from stigmasterol 7 . The Breslow remote functionalization process has been extended to several more highly functionalized steroid derivatives, especially those with oxygen functionality in the B-ring. The key steps of the synthesis of xestobergsterol A 1a and its analogues, 7-deoxyxestobergsterol A 1d and 16,23- seco -23-deoxyxestobergsterol A 73 , are the Breslow remote functionalization of oxygenated steroids and for compounds 1a and 1d , a novel base-catalyzed epimerization-aldol condensation of a dione to give the desired CD- cis ring structure of the xestobergsterols. Thus the known alcohol 75 , prepared from stigmasterol 7 , was taken to the tetraacetate 107 which was then converted via a Breslow remote functionalization into the alkene aldehyde 114 which was transformed in 5 steps to xestobergsterol A 1a . Testing of the synthetic materials showed that the two analogues, 7-deoxyxestobergsterol A 1b and 16,23- seco -23-deoxyxestobergsterol A 73 , are also potent inhibitors of histamine release with IC 50 values (IC 50 500 nM and 750 nM, respectively) being only 1015 times less than that of xestobergsterol A itself (50 nM).

An enantiospecific synthesis of structure 1, previously assigned to the antitubercular marine natural product pseudopteroxazole, has been accomplished as outlined in Scheme 1. Coupling of diene acid 3 and amino phenol 4 produced the amide 5, which was subjected to a novel oxidative intramolecular Diels−Alder reaction to generate the tricyclic lactam 6a stereoselectively. This product was transformed via intermediates 7−11 into the diene 13. Cationic cyclization of 13 afforded two diastereomeric tricyclic amphilectanes which were separated and transformed by parallel four-step sequences into 1 and 2, respectively. Neither 1 nor 2 were identical with pseudopteroxazole, indicating a need for revision of the structure, probably to 16.

An enantiospecific synthesis of structure 1, previously assigned to the antitubercular marine natural product pseudopteroxazole, has been accomplished as outlined in Scheme 1. Coupling of diene acid 3 and amino phenol 4 produced the amide 5, which was subjected to a novel oxidative intramolecular Diels−Alder reaction to generate the tricyclic lactam 6a stereoselectively. This product was transformed via intermediates 7−11 into the diene 13. Cationic cyclization of 13 afforded two diastereomeric tricyclic amphilectanes which were separated and transformed by parallel four-step sequences into 1 and 2, respectively. Neither 1 nor 2 were identical with pseudopteroxazole, indicating a need for revision of the structure, probably to 16.

Revised structures are proposed for pseudopterosin G-J aglycon and helioporin E.

Thermolysis of the 3,5-hexadienyl acrylates (a) proceeds via the transition state E in which the connecting chain adopts a boatlike conformation due to a preference for maintaining ester overlap to afford the products b with good diastereoselectivity.