Supplementary Figure S1. Chemistry/X-ray. Supplementary Figure S2. Biochemical activity of ALK TKIs. Supplementary Figure S3. Kinome profiling of NVL-655. Supplementary Figure S4. Potency in cell viability assay. Supplementary Figure S5. Maximal effect (Emax) in cell viability assay. Supplementary Figure S6. Cellular activity of NVL-655. Supplementary Figure S7. Comparing NVL-655 and lorlatinib potency. Supplementary Figure S8. Pathway analysis in cell lines. Supplementary Figure S9. Further evidence for on-target activity of NVL-655. Supplementary Figure S10. TRK activity. Supplementary Figure S11. Selectivity for ALK versus TRK using additional assays. Supplementary Figure S12. In vivo studies for ALK fusion with a wild-type kinase domain. Supplementary Figure S13. In vivo studies for ALK fusion with G1202R or I1171N single mutations. Supplementary Figure S14. In vivo studies for ALK fusion with G1202R compound mutations. Supplementary Figure S15. Pharmacodynamic analysis in G1202R compound mutation models. Supplementary Figure S16. Body weight and H&E staining for the YU-1077 intracranial study. Supplementary Figure S17. Estimating TRKB inhibition in humans and mice.

NVL-520 Modeling, Properties, Biochemical Activity, Cellular Activity, and In Vivo Activity

Abstract Background: Oncogenic mutations and gene amplifications in the HER2 receptor tyrosine kinase are detected in approximately 2 - 4% and 1 - 5%, respectively, of non-small cell lung cancers (NSCLC) in the US. Exon 20 insertion mutations (exon20ins) are the predominant HER2 mutations in NSCLC, and ~50% of HER2-mutant metastatic NSCLC patients develop brain metastases. The antibody drug conjugate trastuzumab deruxtecan (T-DXd) has received FDA accelerated approval for HER2-mutant NSCLC, but currently there are no approved tyrosine kinase inhibitors (TKIs) for this indication. NVL-330 is a novel, brain-penetrant, HER2-selective TKI, designed to address the combined medical need of treating HER2-mutant tumors, avoiding treatment related adverse events due to off-target inhibition of wild-type EGFR, and treating brain metastases. Here we characterized the preclinical pharmacological profile of NVL-330 in comparison to other investigational HER2-targeting TKIs and T-DXd. Methods: TKIs were profiled in cellular phospho-HER2, phospho-EGFR, and viability assays. In vivo antitumor studies were performed in nude mice with HER2-altered subcutaneous or intracranial tumors. T-DXd concentration was measured in an ELISA assay. The unbound brain-to-plasma partitioning ratio (Κp,uu) was determined at one hour after oral 10 mg/kg dosing in Wistar Han rats. Results: NVL-330 broadly inhibited HER2 oncogenic alterations, including amplified wild-type HER2, HER2 exon20ins and non-exon20ins mutants, with potency and selectivity over wild-type EGFR comparable to investigational HER2-selective TKI zongertinib. By contrast, NVL-330 had a higher Kp,uu than zongertinib in rats. In a patient-derived xenograft model harboring HER2 exon20ins, treatment with NVL-330 suppressed phospho-HER2 and downstream signaling, increased total HER2 and caused dose-dependent tumor regression at well tolerated doses. In the HER2amp NCI-N87 subcutaneous xenograft model, oral administration of 30 mg/kg NVL-330 twice per day induced tumor regression, similar to that achieved by intravenous administration of 10 mg/kg T-DXd once every three weeks, a human-relevant dose in mice. By comparison, in the intracranial setting with the same tumor model and dosing regimens, NVL-330 induced tumor regression, whereas T-DXd provided tumor stasis. Pharmacokinetic analysis in these intracranial tumor bearing mice indicated that NVL-330 had significantly higher brain penetrance than T-DXd. Conclusions: NVL-330 was broadly and selectively active against HER2 oncogenic alterations in vitro and in vivo, and demonstrated brain penetrance in rodents exceeding that of HER2 investigational TKI zongertinib and T-DXd. This preclinical profile supports the potential for NVL-330 to address a medical need for patients with HER2-driven cancers, including those with brain metastases. Citation Format: Yuting Sun, Kristin L. Andrews, Anupong Tangpeerachaikul, Tuan M. Nguyen, Baudouin Gerard, Nancy E. Kohl, Joshua C. Horan, Henry E. Pelish. Preclinical characterization of NVL-330, a selective and brain penetrant HER2 tyrosine kinase inhibitor with broad activity on HER2 oncogenic alterations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1979.

Abstract Three generations of tyrosine kinase inhibitors (TKI) have been approved for anaplastic lymphoma kinase (ALK) fusion–positive non–small cell lung cancer. However, none address the combined need for broad resistance coverage, brain activity, and avoidance of clinically dose-limiting TRK inhibition. NVL-655 is a rationally designed TKI with >50-fold selectivity for ALK over 96% of the kinome tested. In vitro, NVL-655 inhibits diverse ALK fusions, activating alterations, and resistance mutations, showing ≥100-fold improved potency against ALKG1202R single and compound mutations over approved ALK TKIs. In vivo, it induces regression across 12 tumor models, including intracranial and patient-derived xenografts. NVL-655 inhibits ALK over TRK with 22-fold to >874-fold selectivity. These preclinical findings are supported by three case studies from an ongoing first-in-human phase I/II trial of NVL-655 which demonstrate preliminary proof-of-concept clinical activity in heavily pretreated patients with ALK fusion–positive non–small cell lung cancer, including in patients with brain metastases and single or compound ALK resistance mutations. Significance: By combining broad activity against single and compound ALK resistance mutations, brain penetrance, and selectivity, NVL-655 addresses key limitations of currently approved ALK inhibitors and has the potential to represent a distinct advancement as a fourth-generation inhibitor for patients with ALK-driven cancers.

Supplementary Methods, Table 1, Figure Legends 1-5 from MK-2461, a Novel Multitargeted Kinase Inhibitor, Preferentially Inhibits the Activated c-Met Receptor

Abstract ROS1 tyrosine kinase inhibitors (TKI) have been approved (crizotinib and entrectinib) or explored (lorlatinib, taletrectinib, and repotrectinib) for the treatment of ROS1 fusion–positive cancers, although none of them simultaneously address the need for broad resistance coverage, avoidance of clinically dose-limiting TRK inhibition, and brain penetration. NVL-520 is a rationally designed macrocycle with >50-fold ROS1 selectivity over 98% of the kinome tested. It is active in vitro against diverse ROS1 fusions and resistance mutations and exhibits 10- to 1,000-fold improved potency for the ROS1 G2032R solvent-front mutation over crizotinib, entrectinib, lorlatinib, taletrectinib, and repotrectinib. In vivo, it induces tumor regression in G2032R-inclusive intracranial and patient-derived xenograft models. Importantly, NVL-520 has an ∼100-fold increased potency for ROS1 and ROS1 G2032R over TRK. As a clinical proof of concept, NVL-520 elicited objective tumor responses in three patients with TKI-refractory ROS1 fusion–positive lung cancers, including two with ROS1 G2032R and one with intracranial metastases, with no observed neurologic toxicities. Significance: The combined preclinical features of NVL-520 that include potent targeting of ROS1 and diverse ROS1 resistance mutations, high selectivity for ROS1 G2032R over TRK, and brain penetration mark the development of a distinct ROS1 TKI with the potential to surpass the limitations of earlier-generation TKIs for ROS1 fusion–positive patients. This article is highlighted in the In This Issue feature, p. 517

Supplementary Methods from Lung Cancer Cell Lines Harboring MET Gene Amplification Are Dependent on Met for Growth and Survival

Supplementary Figure 3 from MK-2461, a Novel Multitargeted Kinase Inhibitor, Preferentially Inhibits the Activated c-Met Receptor

Supplementary Information from Lung Cancer Cell Lines Harboring MET Gene Amplification Are Dependent on Met for Growth and Survival

Supplementary Figure 1, Table 1 from De novo Discovery of a γ-Secretase Inhibitor Response Signature Using a Novel In vivo Breast Tumor Model

Supplementary Figures 1-5, Tables 1-2 from Inhibition of NOTCH Signaling by Gamma Secretase Inhibitor Engages the RB Pathway and Elicits Cell Cycle Exit in T-Cell Acute Lymphoblastic Leukemia Cells

Supplementary Methods and Figure Legends

Supplementary Table S1 from Identification of biomarkers for tumor endothelial cell proliferation through gene expression profiling

Supplemental Figure 1. Bioluminescence plots of individual animals following treatment with immune checkpoint blocking mAbs. Supplemental Figure 2. Immune cell infiltrates within intracranial GL261-luc2 tumor following treatment with isotype control IgG or combination therapy with CTLA-4 plus PD-1 mAb therapy. Supplemental Figure 3: Treatment antibody and detection antibody for PD-1 do not share same the epitope. Supplemental Figure 4. Representative immunohistochemistry results of intratumoral CD3+, CD4+ and CD8+ T cell infiltrates following immune checkpoint therapy with mAbs against CTLA-4, PD-1 and both CTLA-4 and PD-1. Supplemental Figure 5. Immune cell infiltration within the draining cervical lymph node (cLN) of GL261-luc2 tumor-bearing mice following treatment with isotype control IgG or combination therapy with mAbs against CTLA-4 plus PD-1. Supplemental Figure 6. Comparisons of chemokine expression levels in sera of mice treated with checkpoint blockade. Supplemental Figure 7: Expression of checkpoint ligands and receptors on GL261-Luc cells in vitro. Supplementary Table 1. Arrangement of chemokines on the Mouse Chemokine Antibody Array.

Supplementary Figure 1 Legend from Lung Cancer Cell Lines Harboring MET Gene Amplification Are Dependent on Met for Growth and Survival

Abstract Aberrant signaling through the fibroblast growth factor 19 (FGF19)/fibroblast growth factor receptor 4 (FGFR 4) signaling complex has been shown to cause hepatocellular carcinoma (HCC) in mice and has been implicated to play a similar role in humans. We have developed BLU9931, a potent and irreversible small-molecule inhibitor of FGFR4, as a targeted therapy to treat patients with HCC whose tumors have an activated FGFR4 signaling pathway. BLU9931 is exquisitely selective for FGFR4 versus other FGFR family members and all other kinases. BLU9931 shows remarkable antitumor activity in mice bearing an HCC tumor xenograft that overexpresses FGF19 due to amplification as well as a liver tumor xenograft that overexpresses FGF19 mRNA but lacks FGF19 amplification. Approximately one third of patients with HCC whose tumors express FGF19 together with FGFR4 and its coreceptor klotho β (KLB) could potentially respond to treatment with an FGFR4 inhibitor. These findings are the first demonstration of a therapeutic strategy that targets a subset of patients with HCC. Significance: This article documents the discovery of BLU9931, a novel irreversible kinase inhibitor that specifically targets FGFR4 while sparing all other FGFR paralogs and demonstrates exquisite kinome selectivity. BLU9931 is efficacious in tumors with an intact FGFR4 signaling pathway that includes FGF19, FGFR4, and KLB. BLU9931 is the first FGFR4-selective molecule for the treatment of patients with HCC with aberrant FGFR4 signaling. Cancer Discov; 5(4); 424–37. ©2015 AACR. See related commentary by Packer and Pollock, p. 355 This article is highlighted in the In This Issue feature, p. 333

Supplementary Figure S1. Potency against FGFR family and kinome selectivity. Supplementary Figure S2. Kinases with a cysteine at the equivalent position to Cys552 in FGFR4. Supplementary Figure S3. MALDI Mass Spectrometry of FGFR4. Supplementary Figure S4. Induction of Caspase 3/7 activity in Hep 3B cells. Supplementary Figure S5. FGFR4 turnover rates in Hep 3B cells. Supplementary Figure S6. Analysis of TCGA HCC samples. Supplementary Table S1. Kinetic parameters of FGFR4 inhibition by BLU9931. Supplementary Table S2. Pharmacokinetic parameters for BLU9931.

Supplementary Materials and Methods Supplementary References Tables S1 to S3 and legends Figures S1 to S4 and legends

Supplementary Figures 1-4 from PDK1 Attenuation Fails to Prevent Tumor Formation in PTEN-Deficient Transgenic Mouse Models

Abstract ROS1 is a proto-oncogene that encodes the receptor tyrosine kinase ROS1, which can be aberrantly activated by gene rearrangement to drive tumor cell proliferation, survival, and metastasis. In non-small cell lung cancer (NSCLC), ROS1 rearrangements are detected in up to 3% of patients with up to 40% of these patients also presenting with accompanying central nervous system (CNS) metastases. Although various tyrosine kinase inhibitors (TKI) have been approved or are under development for treating ROS1-positive patients, these therapies are associated with one or more of the following significant challenges: 1. emergence of ROS1 mutations that confer disease resistance, including the G2032R solvent front mutation, 2. disease progression into the CNS, and 3. treatment-related adverse events (AEs) associated with off-target kinase inhibition, notably TRKB in the CNS. NVL-520 was developed to address the challenges listed above. Preclinical studies have shown that NVL-520 inhibits wild-type and drug resistance mutants of the ROS1 kinase and demonstrated activity against CNS disease with minimal kinase off-target activity, suggesting an opportunity for durable responses for patients with ROS1-positive disease. Here we report detailed characterization of cellular and antitumor activity of NVL-520 in patient-derived models of ROS1-driven NSCLC. Methods: Cellular and antitumor activity of NVL-520 were evaluated in patient-derived models of NSCLC representing ROS1 and ROS1-G2032R driven disease. For in vivo studies, treatments were administered orally. Western blotting, immunohistochemistry, and gene expression analyses were used to measure pharmacodynamic (PD) effects in tumor tissue. Pharmacokinetic (PK) analyses were also performed. Results: In patient-derived cell (PDC) models of EZR-ROS1 and CD74-ROS1 G2032R, NVL-520 treatment resulted in antiproliferative activity, with IC50 values < 10 nM, and reduced levels of phospho-ROS1 and markers of downstream signaling. Studies in patient-derived xenograft (PDX) models demonstrated that NVL-520 was well-tolerated and resulted in dose-dependent antitumor activity, including tumor regression in all models. Analysis of PD biomarkers showed that NVL-520 suppressed ROS1 phosphorylation and significantly impacted ROS1-dependent signaling pathways. PK analysis demonstrated dose-dependent differences in NVL-520 exposure. PD-Efficacy correlation analysis showed that changes in PD were consistent with dose-dependent antitumor activity of NVL-520 with distinct changes observed at doses that induce tumor stasis versus tumor regression. Conclusion: NVL-520 exhibits antiproliferative and antitumor activity in NSCLC models driven by ROS1 and ROS1-G2032R solvent front mutation. We believe the findings presented here support evaluation of NVL-520 for the treatment of patients with ROS1-driven disease. Citation Format: Amit M. Deshpande, Satoshi Yoda, Anupong Tangpeerachaikul, Nancy E. Kohl, Joshua C. Horan, Aaron N. Hata, Henry E. Pelish. Preclinical antitumor activity of NVL-520 in patient-derived models harboring ROS1 fusions, including G2032R solvent front mutation [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P249.