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Exon 20 insertion mutations (Ex20ins) are the third most common type of EGFR oncogenic mutation in lung cancer patients. While several small molecules have been approved (mobocertinib) or are in clinical development (e.g. CLN-081, BDTX-189) none have demonstrated meaningful CNS activity and they can be associated with treatment-limiting adverse events, including wild-type (WT) EGFR-mediated toxicities. LNG-451 is a CNS-penetrant, wild-type EGFR-sparing, covalent inhibitor of EGFR Ex20ins. In cell viability assays dependent on five EGFR Ex20ins mutations, LNG-451 was a potent inhibitor (IC50 7 - 78 nM) including the three most prevalent insertions (V769_D770insASV IC50 = 78 nM, D770_N771insSVD IC50 = 53 nM, H773_V774insNPH IC50 = 75 nM). LNG-451 had low potency in WT EGFR dependent cell assays (WT EGFR Ba/F3 IC50=1,960 nM, cell panel IC50 = 1630 nM). LNG-451 was 21-fold selective for its least potent Ex20ins (V769_D770insASV) relative to WT EGFR. LNG-451 potently inhibited cells dependent on EGFR L861Q (IC50 = 6 nM), EGFR G719S (IC50 = 8 nM), and EGFR G719S/T263P (IC50 = 11 nM). LNG-451 is a highly selective kinase inhibitor (1 μM LNG-451, >90% inhibition for 7 of 409 WT kinases) with screening hits having a similarly positioned cysteine as EGFR. In a Ba/F3 cell-derived xenograft model harboring an EGFR V769_D770insASV exon 20 insertion mutation (EGFR V769_D770insASV CDX), 10 and 50 mg/kg LNG-451 QD PO dosing for 15 days was well-tolerated and resulted in tumor growth inhibition (TGI) of 75.7% and 105.4%, respectively. LNG-451 was efficacious and well-tolerated in the EGFR Ex20ins HuPrime® LU0387 H773-V774insNPH patient-derived xenograft tumor model: 10 mg/kg LNG-451 QD, 67% TGI; 50 mg/kg LNG-451 QD, 91% tumor regression. LNG-451 was highly active in the intracranial PC9-luc xenograft mouse model. In a single dose PK/PD study using the EGFR V769_D770insASV CDX, similar LNG-451 concentrations were observed in tumor, large intestine, and skin tissues (e.g. 50 mg/kg LNG-451 3h post dose: tumor, 8387 ng/mL; large intestine tissue, 14483 ng/mL; skin, 4623 ng/mL). LNG-451 potently suppressed EGFR phosphorylation in tumor tissue (e.g. 99%, 3 h post 50 mg/kg dose) but had minimal-to modest suppression of phospho-EGFR in large intestine tissue (e.g. 4.2%, 3h post 50 mg/kg dose) and skin tissue (e.g 1.9%, 3h post 50 mg/kg dose). Taken together, LNG-451 is a wild-type EGFR-sparing, CNS-penetrant EGFR Ex20ins inhibitor that is expected to provide strong anti-tumor efficacy for patients with advanced/metastatic solid cancers harboring oncogenic EGFR exon 20 insertions with reduced WT EGFR-driven toxicities. Citation Format: Brion W. Murray, Anjali Pandey, Bruce Roth, Tracy Saxton, Daniel J. Estes, Himanshu Agrawal, Santosh Vishwakarma, Gurulingappa Hallur, Ishtiyaque Ahmad, Ravi Trivedi, Helen Jenkins, Paul G. Pearson. LNG-451 is a potent, CNS-penetrant, wild-type EGFR sparing inhibitor of EGFR exon 20 insertion mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3332.

Exon 20 insertion mutations (Ex20ins) are the 3rd most common type of EGFR oncogenic mutation in lung cancer patients and are often associated with brain metastases. While several small molecules have been approved (mobocertinib) or are in clinical development (e.g. CLN-081, BDTX-189) none have demonstrated meaningful CNS activity and they can be associated with treatment-limiting adverse events, including wild-type (WT) EGFR-mediated toxicities. LNG-451 is a potent covalent inhibitor of EGFR Ex20ins (IC50 7 - 78 nM) and other EGFR oncogenic mutations (e.g L858R IC50 = 3 nM, exon 19 deletion IC50 = 24 nM) while being selective against WT EGFR (IC50 = 1630 nM). LNG-451 causes tumor regression in both cell line-derived and patient-derived EGFR Ex20ins-dependent xenograft tumor models. In a rat CNS PK model, a single oral dose of 30 mg/kg LNG-451 achieved high exposures in plasma (AUC0 - 8h = 9771 ng•h/mL) and brain (AUC0 - 8h = 10,041 ng•h/g). In a rat steady-state CNS PK model, unbound partition coefficient for LNG-451 was determined to be Kpu,u = 0.66. To evaluate the potential for LNG-451 to treat metastatic brain disease driven by EGFR mutations, an intracranial PC9-luc xenograft mouse model harboring an EGFR exon 19 deletion mutation was used (LNG-451 cellular EGFR Del19 IC50 = 10.6 nM). LNG-451 had dose-dependent exposures in the plasma and brain with both high mean brain exposure (e.g 1074 ng/g with 25 mg/kg LNG-451) and high mean brain to plasma ratios (e.g. 0.451 for 25 mg/kg LNG-451). LNG-451 treatments were well-tolerated and produced significant anti-tumor activities with 99.5% (2.5 mg/kg LNG-451) and 99.9% (25 mg/kg LNG-451) reductions in total bioluminescence signal (BLI) and regression of tumor BLI (2.5 mg/kg LNG-451 28.8% regression; 25 mg/kg LNG-451, 80.3% regression). An ex vivo imaging analysis of the brain and spinal cords from all animals at the end of the study showed a dose-dependent decrease in the bioluminescent signal in both brain and spinal cords from mice treated with LNG-451 relative to the vehicle control animals. Taken together, LNG-451 is a CNS-penetrant, wild-type EGFR-sparing, EGFR Ex20ins inhibitor that is expected to provide strong anti-tumor efficacy for patients with advanced/metastatic solid cancers harboring oncogenic EGFR exon 20 insertions with reduced WT EGFR driven toxicities. Citation Format: Paul G. Pearson, Anjali Pandey, Bruce Roth, Tracy Saxton, Daniel J. Estes, Ravi Trivedi, Himanshu Agrawal, Gurulingappa Hallur, Ishtiyaque Ahmad, Helen Jenkins, Brion W. Murray. LNG-451, a potent inhibitor of EGFR exon 20 insertion mutations with high CNS exposure [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3261.

This abstract was withdrawn by the authors. Citation Format: Dann S, Chionis J, Eisele K, Zhang Q, Liu C, Yuan J, Miller N, Murray B, Xu M, Solowiej J, Wei P, Weinrich S, Sutton S, Behenna D, Ninkovic S, Hoffman R, Freeman-Cook K, Jessen B, Huser N, Zhang C, Visswanathan R, Boras B, VanArsdale T, White MA. Withdrawn [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P6-20-06.

NTRK chromosomal rearrangements yield oncogenic TRK fusion proteins that are sensitive to first-generation TRK inhibitors (larotrectinib, entrectinib) but the emergence of NTRK mutations limits their efficacy. Next-generation TRK inhibitors (repotrectinib, selitrectinib) have compact macrocyclic structures designed to limit the susceptibility to mutations. However, a detailed understanding of mutant potency and precise binding interactions are lacking. TRK inhibitors were evaluated in cellular models expressing TRKA/B/C fusions with wild-type (WT) and resistance mutations: solvent front (SFM), gatekeeper (GKM), activation loop (xDFG), and compound mutations. In cell proliferation assays, differential potencies against wild type TRKA/B/C fusions were observed: larotrectinib (IC50 23.5 - 49.4 nM), entrectinib (IC50 0.3 - 1.3 nM), selitrectinib (IC50 1.8 - 3.9 nM), and repotrectinib (IC50 < 0.2 nM). First generation TRK inhibitors with extended structures had reduced potency against resistance mutations. Larotrectinib had minimal activity (IC50 >600 nM) against all TRK mutations and entrectinib had >400-fold decreased against all mutations except GKM where there was a range of potencies (IC50 < 0.2 - 60.4 nM). Repotrectinib and selitrectinib were less affected by resistance mutations however repotrectinib was approximately 10-fold more potent than selitrectinib against SFM and compound mutations and 100-fold more potent against GKM. For TRKA/B/C xDFG mutations, repotrectinib had moderate potency (IC50 11.8 - 67.6 nM) while selitrectinib was less potent (IC50 124 - 341 nM). Co-crystal structures of repotrectinib with TRKA and TRKA harboring a SFM provide insight into how subtle differences in macrocyclic inhibitor structure and conformational profiles affect potency and mutant selectivity. Analysis of the first protein structure of a kinase harboring a common SFM (TRKA G595R) revealed unexpected intramolecular interactions which provide insight into its prevalence. Repotrectinib, but not selitrectinib, caused tumor regression in LMNA-NTRKA xenograft models harboring GKM, SFM, or GKM+SFM compound mutations. In the clinic, tumor regression was observed with repotrectinib treatment of a larotrectinib-resistant cholangiocarcinoma patient with both LMNA-TRKA GKM and SFM mutations. Repotrectinib has shown responses in patients with TRK driven tumors with or without resistant mutations in the ongoing global registrational TRIDENT-1 study and has been granted 3 fast track designations. Taken together, the current data characterizes TRK inhibitor potency against resistance mutations and highlights structural characteristics of repotrectinib that enable potent inhibition of TRK proteins and evasion of drug resistance mediated by TRK mutations. Citation Format: Alexander Drilon, Dayong Zhai, Evan Rogers, Wei Deng, Xi Chen, Paul Sprengeler, Siegfried H. Reich, Brion W. Murray. Molecular characteristics of repotrectinib that enable potent inhibition of TRK fusion proteins and broad mutant selectivity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1119.

Within the tumor microenvironment (TME), CSF1R (colony stimulating factor 1 receptor) signaling plays a pivotal role in recruitment and enrichment of M2 tumor-associated macrophages (TAM) that dampen the anti-tumor immune response and promote tumor progression. Although CSF1R inhibitors have shown promise in the clinic, tumor cells and TAMs can bypass CSF1R inhibition and expand M2 TAMs through other growth factor receptors, such as MET (hepatocyte growth factor receptor) to confer resistance to targeted therapies and immune checkpoint inhibitors. Through autocrine signaling pathways, CSF1R and MET activation in tumors contribute to tumor invasiveness and metastasis. Therefore, inhibitors that target both CSF1R and MET, such as TPX-0022 (a compact macrocyclic inhibitor of MET/SRC/CSF1R), may more effectively suppress CSF1R mediated signaling in tumor cells and the stroma. In the current study, the role of TPX-0022 in the anti-tumor immune response is evaluated in the context of both normal immune cells and immunocompetent syngeneic murine models. TPX-0022 blocks both HGF and CSF1 mediated signaling and proliferation of MET and CSF1R dependent cell lines. In the MC-38 syngeneic model, TPX-0022 treatment repolarized TAMs towards M1 anti-tumorigenic macrophages, increased CD8 T cells, and inhibited tumor growth. In a mixed lymphocyte reaction assay, we demonstrated that pretreatment of macrophages with TPX-0022 enhances macrophage function in driving the activation and proliferation of CD8 T cells. Furthermore, TPX-0022 treatment of anti-C3/CD28 and or SIINFEKL peptide stimulated murine T cells had no deleterious effects on T cell activation and proliferation. Additionally, TPX-0022 enhanced T cell cytotoxic function towards cancer cells in a dose-dependent manner. Detailed analysis of the TME in CT-26 animals treated with TPX-0022 showed a significant reduction in TAMs, rebalancing both immunosuppressive Treg and M2 macrophages towards anti-tumorigenic CTL (CD8+ CD69+ GnzB+) and M1 macrophages. The combination of TPX-0022 and a PD-1 antibody resulted in significant downregulation of pro-tumorigenic cytokines IL-10, IP-10, IL-1β, and HGF, without impacting the anti-tumor cytokine IFN-γ. In the MC-38 model, TPX-0022 demonstrated single-agent activity and a trend to enhanced efficacy in combination with a PD-1 antibody. Taken together, the preclinical data demonstrates that the MET/SRC/CSF1R inhibitor TPX-0022 can reprogram tumor associated macrophages as well as support antigen presentation and activation of T cells within the TME. A TPX-0022 combination with a PD-1 therapy yielded more anti-tumor activity than single agent treatments. Together, the in vitro and in vivo preclinical data provide a mechanistic rationale for the potential utility of TPX-0022 in combination with immune checkpoint inhibitors. Citation Format: Vikas K. Goel, Wei Deng, Dayong zhai, Nathan V. Lee, Ana Parra, Brion W. Murray. TPX-0022, a potent MET/SRC/CSF1R inhibitor that modulates the tumor immune microenvironment in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1444.

Three generations of ALK inhibitors are approved for the treatment of ALK+ NSCLC but their efficacy is often limited by ALK resistance mutations. The solvent front mutation G1202R and gatekeeper mutation L1196M are major resistance mechanisms to the first two generations of inhibitors. Patients treated with second generation inhibitors are reported to progress with multiple mutations on separate alleles (mutations in trans). In contrast, 35 - 48% of patients treated with lorlatinib progress with multiple mutations on the same allele (compound mutations, mutations in cis). TPX-0131 is an ALK inhibitor with a compact macrocyclic structure designed to bind completely within the ATP binding boundary and overcome a spectrum of single and compound ALK resistant mutations. TPX-0131 was profiled against previous generations of ALK inhibitors both in vitro and in vivo. In biochemical assays, TPX-0131 potently inhibits (IC50 100-fold, crizotinib). TPX-0131 potently inhibits EML4-ALK harboring a G1202R solvent front mutation (IC50 = 0.2 nM) which is >100-fold more potent than previous generations of ALK inhibitors. TPX-0131 potently inhibits ALK harboring a gatekeeper mutation (IC50 = 0.5 nM) and is >10-fold more potent than previous generations of ALK inhibitors. TPX-0131 potently inhibits ALK with a L1198F hinge area mutation (IC50 = 0.2 nM) which is 87 - 3000-fold more potent than previous generations of ALK inhibitors. TPX-0131 is the most potent inhibitor against nine EML4-ALK double and triple compound mutations (6 with IC50 < 1 nM, 3 with IC50 1.6 - 14.9 nM). Evaluation of ALK phosphorylation as a pharmacodynamic marker in tumors showed potent ALK inhibition by TPX-0131 that correlated with TPX-0131 exposure. In Ba/F3 cell-derived xenograft tumor models with EML4-ALK mutations, TPX-0131 (2, 5, 10 mg/kg BID) demonstrated robust anti-tumor activity in the G1202R model (64%, 120%, 200% TGI), G1202R/L1198F model (complete regression, all doses), and G1202R/L1196M model (44%, 83% and 200% TGI). In contrast, lorlatinib (5 mg/kg BID) caused 31% TGI in the G1202R/L1198F model and did not have statistically significant TGI in the G1202R/L1196M model. Taken together, TPX-0131 is a next generation ALK inhibitor that has preclinical potency against WT ALK as well as a broad spectrum of acquired resistance mutations, especially compound mutations, which currently lack any effective ALK inhibitor therapy. Citation Format: Brion W. Murray, Dayong Zhai, Wei Deng, Evan Rogers, Xin Zhang, Jane Ung, Vivian Nguyen, Han Zhang, Maria Barrera, Ana Parra, Jessica Cowell, Dong Lee, Herve Aloysius. TPX-0131, a potent inhibitor of wild type ALK and a broad spectrum of both single and compound ALK resistance mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1469.

KRAS is frequently mutated in multiple cancer types. Therapeutic targeting of KRAS has proven challenging until recent success of KRAS-G12C inhibitor AMG510 that demonstrated tumor regression in lung and colon cancer patients with a KRAS-G12C mutation. However, resistance via feedback reactivation or development of a bypass are expected to limit anti-tumor efficacy of KRAS-G12C inhibitors, favoring a combination approach. Adaptive upregulation of the PI3K/AKT signaling pathway has been recognized as a common resistance mechanism that promotes cancer cell survival and may temper efficacy of KRAS-G12C inhibitors. SRC/FAK signaling promotes tumor cell survival by activation of the PI3K/AKT survival pathway supporting concomitantly targeting SRC/FAK as a promising strategy to potentiate KRAS G12C inhibitors. In addition, oncogenic KRAS induces secretion of various cytokines and growth factors leading to modulation of the tumor microenvironment to promote tumor growth. Targeting the cytokine secretion pathway via JAK2/STAT3 modulation may be important to overcome the resistance to direct KRAS inhibition. Therefore, combinations of KRAS inhibitors with agents that target apoptotic processes and feedback reactivation may represent a promising therapeutic approach. Repotrectinib, a next generation ROS1/TRK/ALK inhibitor, also inhibits SRC/FAK/JAK2 at therapeutically relevant concentrations. Here, we present synergistic effects of repotrectinib in combination with a KRAS-G12C inhibitor in KRAS-G12C mutant cancer models via simultaneous inhibition of SRC/FAK/JAK2. Adding repotrectinib to AMG510 improves AMG510 effectiveness on KRAS-G12C mutant cancer cells with notably increased apoptosis. For example, repotrectinib (1 μM) shifted the antiproliferation IC50 of AMG510 from 213 nM to 3 nM in KRAS-G12C mutant H358 cells. Treatment of H358 cells with AMG510 alone (100 nM) or repotrectinib alone (1 µM) resulted in slight increases in cleaved PARP and caspase-3 proteins, whereas larger increases were observed with the combination. Although high levels of phosphorylated ERK, AKT, STAT3 and FAK were detected in H358 cells, AMG510 (100 nM) was only able to partially modulate phospho-ERK (pERK) with no modulation on pAKT, pSTAT3 and pFAK and repotrectinib (1 μM) suppressed pSTAT3 and pFAK with minimum modulation on pERK and pAKT. The combination in H358 cells results in stronger suppression of pERK and pAKT than either single agent alone, which is consistent with the observed increase in activation of apoptosis. Additional combination data in KRAS-G12C mutant cancer models will be presented. Overall, these studies warrant further clinical investigation on the combination of KRAS-G12C inhibitors with repotrectinib in patients with KRAS-G12C mutation for potential response and duration improvement of current investigational KRAS-G12C inhibitors. Citation Format: J. Jean Cui, Dayong Zhai, Wei Deng, Laura Rodon, Nathan Lee, Brion Murray. Repotrectinib increases effectiveness of KRAS-G12C inhibitors in KRAS-G12C mutant cancer models via simultaneous SRC/FAK/JAK2 inhibition [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1958.

Kirsten Rat Sarcoma Viral Oncogene homolog (KRAS) is the most frequently mutated oncogene in a broad spectrum of human cancers, including ~30% of non-small cell lung cancer (NSCLC), ~45% of colorectal cancer (CRC) and ~75% of pancreatic cancer. The MEK inhibitor trametinib, which targets the primary downstream signaling pathway activated by mutant KRAS, has shown limited activity when used as a single agent in patients with previously treated KRAS-mutant positive NSCLC. Compensatory upregulation of the PI3K/AKT survival signaling and intrinsic or treatment induced epithelial-to-mesenchymal transition (EMT) are among resistance mechanisms of MEK inhibition in KRAS mutant cancers. SRC/FAK signaling modulates the PI3K/AKT survival pathway and is involved in the integrin mediated EMT signaling. In addition, MEK inhibition has been reported to activate the JAK2/STAT3 pathway to evade therapy. Therefore, the combination of a MEK inhibitor with agents that simultaneously inhibit SRC, FAK and JAK2 may represent a promising therapeutic approach to effectively target KRAS mutant cancers. Repotrectinib, a next generation ROS1/TRK/ALK investigational drug, also inhibits SRC/FAK/JAK2 at therapeutically relevant concentrations. Here, we present synergistic effects of repotrectinib in combination with trametinib in KRAS mutant cancer models. Increased anti-proliferative activity was demonstrated by the combination across panels of KRAS mutant NSCLC and CRC cell lines that harbor a spectrum of KRAS mutations. Additionally, the combination of trametinib and repotrectinib in KRAS mutant cell lines caused an enhanced induction of apoptosis in comparison with trametinib or repotrectinib alone. Trametinib induced a rebound in phospho-AKT (pAKT) levels in a subset of KRAS mutant cells which was substantially suppressed by the combination with repotrectinib. In contrast, suppression of the trametinib-induced pAKT rebound was not achieved by combinations of trametinib with dasatinib (SRCi), ruxolitinib (JAK1/2i), or defactinib (FAKi), suggesting that concomitant SRC/FAK/JAK2 inhibition is potentially necessary. Combination of trametinib and repotrectinib in KRAS mutant murine xenograft studies resulted in suppression of SRC/FAK/STAT3/EGFR/ERK phosphorylation and the enhancement of tumor apoptosis. Importantly, the combination of repotrectinib and trametinib demonstrated increased in vivo anti-tumor effect compared to trametinib alone. Our data suggest that the combination of repotrectinib with trametinib may sustainably suppress the mutant KRAS network signaling to achieve more potent and durable anti-tumor efficacy, and that further clinical investigation of the combination treatment of a MEK inhibitor with repotrectinib in patients with KRAS mutant cancers is warranted. Citation Format: Brion Murray, Wei Deng, Dayong Zhai, Laura Rodon, Nathan Lee, J. Jean Cui. Repotrectinib increases effectiveness of MEK inhibitor trametinib in KRAS mutant cancer models via simultaneous SRC/FAK/JAK2 inhibition [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1957.

The T315I gatekeeper mutation confers resistance to majority of approved ABL1 inhibitors, with only ponatinib demonstrating efficacy in BCR-ABL1(T315I)-driven disease. However, vascular adverse events and selection of resistant compound mutations limit its clinical utility. Hence, there is an unmet need for novel therapies for patients with gatekeeper-mutated Ph+ leukemia. In this study we integrated comprehensive drug sensitivity testing with structural analysis to characterize the tyrosine kinase inhibitor axitinib as a putative novel therapy for BCR-ABL1(T315I)-driven leukemias. To address this we profiled BCR-ABL1(T315I)-driven CML/Ph+ ALL patient samples against 300 anti-cancer compounds (approved and investigational drugs). Ex vivo drug sensitivity testing of primary cells derived from a Ph+ ALL patient revealed a marked and cancer-selective response to the VEGFR inhibitor axitinib. Strikingly, axitinib exhibited higher sensitivity in T315I positive Ph+ patient samples in comparison to T315I negative CML and ALL patient samples. In line with the ex vivo drug response data, axitinib inhibited the kinase activity of ABL1(T315I) with similar potency as its primary target VEGFR2, while the potency to non-mutated ABL1 was 30-fold lower. Analogously, in engineered Ba/F3 cells, axitinib showed a 10-fold higher inhibition of T315I than non-mutated ABL1-driven autophosphorylation and cell growth. To better understand the molecular mechanisms of the BCR-ABL1(T315I) selectivity, we solved the crystal structure of axitinib in complex with ABL1(T315). The structure revealed that axitinib bound to a mutation induced active conformation of ABL1(T315I), different than the binding mode in non-mutated ABL1, likely explaining the increased potency towards ABL1(T315I). Moreover, axitinib occupied a distinct binding space than all approved ABL1 inhibitors, signifying that axitinib will have a unique mutation vulnerability profile. Compassionate two week treatment of a CML patient harboring the T315I mutation with axitinib, resulted in a 5-fold reduction of T315I transcript levels in the bone marrow, further suggesting that axitinib can produce specific and effective responses in patients with BCR-ABL1(T315I)-driven leukemia. In conclusion, we demonstrate that axitinib potently inhibits BCR-ABL1(T315I) via a gatekeeper mutant-selective mechanism. Since axitinib is in clinical use for treatment of refractory renal cell carcinoma with a manageable safety profile, our data provide a sound basis for readily repurposing axitinib for BCR-ABL1(T315I)-driven leukemia. Finally, the distinct mechanism of inhibition by axitinib serves as an exemplar for development of even more effective gatekeeper-mutant selective inhibitors targeting ABL1 as well as other clinically important kinases, such as EGFR and KIT. Citation Format: Tea Pemovska, Eric Johnson, Mika Kontro, Gretchen A. Repasky, Jeffrey Chen, Peter Wells, Ciarán N. Cronin, Michele McTigue, Olli Kallioniemi, Kimmo Porkka, Brion W. Murray, Krister Wennerberg. Axitinib targets gatekeeper-mutant BCR-ABL1(T315I)-driven leukemia in a distinct and selective fashion. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 676. doi:10.1158/1538-7445.AM2015-676

Activating mutations in EGFR confer constitutive activity providing the oncogenic drive in EGFR-mutant NSCLC. First and 2nd generation EGFR tyrosine kinase inhibitors (TKIs) are effective drugs in this setting, but are constrained by dose-limiting toxicities attributed to inhibition of wild type (WT) EGFR and by drug resistance caused, in the majority of cases, via a T790M secondary mutation in EGFR. We report the pharmacology of a novel irreversible 3rd generation EGFR TKI active against EGFR with activating and T790M mutations, but sparing WT EGFR. Our novel 3rd generation EGFR TKI was studied in a variety of in vitro and in vivo models to determine its inhibitory potencies on different EGFR variants, pharmacokinetics (PK), antitumor efficacy, exposure-response relationships, mechanism of action, and predicted human efficacious dose. In enzyme and cell assays, our compound is a highly potent inhibitor of EGFR double mutants (L858R/T790M and Del/T790M) and EGFR activating mutants (L858R and Del), but a weak inhibitor of WT EGFR (26-fold margin over mutant target potencies). Effects on downstream signaling and function indicate the underlying mechanism of the compound is direct inhibition of EGFR, with subsequent inhibition of downstream signaling that results in apoptosis and viable cell decline. In xenograft mouse models, the compound demonstrates tumor growth inhibition and regression at well-tolerated doses in models driven by EGFR double mutants and EGFR activating mutants. The antitumor efficacy is dose-dependent and strongly correlates with inhibition of EGFR phosphorylation and EGFR-mediated downstream signaling, and induction of apoptosis. Plasma concentrations assumed to be sufficient for efficacy (Ceff) were defined using a mathematical model incorporating the plasma levels of the compound, the associated inhibitory effects on EGFR phosphorylation, and the antitumor efficacy in the double and activating mutant xenograft models. Ceff was in agreement across several models and was used with in vitro human PK properties to calculate required human dose. While our compound possesses a similar profile as other recently disclosed 3rd generation EGFR TKIs, this molecule is distinguished by better potency on the activating mutants and by the widest potency margin on WT EGFR. Given that the target potencies and WT margins of 3rd generation EGFR TKIs have been sufficient for tolerated clinical efficacy in preliminary results, it can be inferred that our compound will have similar promise in the clinic. These results support our compound as a novel EGFR TKI with an inhibitory profile and favorable drug-like properties that suggest utility for treating patients with NSCLC with EGFR activating and resistance mutations. Citation Format: Mike Zientek, Sangita Baxi, Henry Cheng, Valeria Fantin, Jun Li Feng, Allison Given, Zelanna Goldberg, Jie Guo, Michelle Hemkens, John Kath, Jennifer Lafontaine, Gary Li, Pramod Mehta, Brion Murray, Sajiv Nair, Simon Planken, Chad Ray, Yuli Wang, Manli Shi, Anand Sistla, Tod Smeal, Greg Stevens, Wei Tan, Paolo Vicini, Marlena Walls, Liu Yang, Min-Jean Yin, Scott L. Weinrich. Characterization of a novel irreversible third generation EGFR TKI that targets T790M-mediated resistant EGFR-mutant NSCLC while sparing wild type EGFR. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2594. doi:10.1158/1538-7445.AM2015-2594

Anaplastic lymphoma kinase (ALK), when aberrantly regulated through gene translocations or mutations, has been implicated as an oncogenic driver in a variety of cancers. XALKORI (crizotinib) is an FDA-approved therapy for locally advanced or metastatic (ALK)-positive non-small cell lung cancer (NSCLC). Mutations of certain residues within the ALK kinase domain have been associated with crizotinib resistance. To understand the molecular basis of mutational resistance, we have kinetically and structurally characterized several clinical mutations of ALK kinase domain. In the basal-state (nonphosphorylated) proteins, mutations C1156Y, F1174L, L1152R and L1196M resulted in 14 to 52-fold increase in catalytic efficiency of phosphorylation of an activation loop peptide. Accordingly, these mutants were more rapidly autophosphorylated, compared to wild-type enzyme. In addition, F1174L, C1156Y and L1156Y had 2 to 6-fold higher catalytic efficiencies when fully activated by autophosphorylation. Conversely, catalytic efficiencies of nonphosphorylated and phosphorylated G1269A variant decreased by 1.2 and 1.6-fold, respectively. Inhibition of L1152R, L1196M and G1269A by crizotinib was reduced by 3-28-fold, compared to wild-type enzyme, from Ki determinations. From direct binding studies of the L1196M “gatekeeper” mutation, the loss of crizotinib binding occurred 6 and 13-fold for phosphorylated and nonphosphorylated proteins, respectively. By crystallographic studies apoenzyme and crizotinib complexes of ALK kinase domain displayed an inactive kinase conformation that is stabilized by an extended hydrophobic network of residues. F1174 and L1196 are part of this hydrophobic core, and mutations of these residues are predicted to destabilize the inactive ALK conformation. Taken together, these results suggest that most of the resistant mutations (e.g. L1196M, F1174L, C1156Y, L1152R) result in a more dynamic protein that increases substrate turnover. In addition, the reduction of crizotinib binding for the mutations in the vicinity of the inhibitor binding site (L1196M, G1269A) is likely a contributing factor for the resistance. This structural and kinetic analysis of mutational resistance may be useful for design of new inhibitors targeting multiple clinical mutations of ALK. Citation Format: Sergei Timofeevski, Wei Liu, Ya-Li Deng, Alexei Brooun, Simon Bergqvist, Shannon Karlicek, Brion Murray, Ben Bolaños, Michele McTigue. Biochemical and structural characteristics of crizotinib-resistant ALK mutants. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4456. doi:10.1158/1538-7445.AM2013-4456