The ALK/ROS1 Inhibitor PF-06463922 Overcomes Primary Resistance to Crizotinib in ALK-Driven Neuroblastoma.
ABSTRACT: UNLABELLED:Neuroblastomas harboring activating point mutations in anaplastic lymphoma kinase (ALK) are differentially sensitive to the ALK inhibitor crizotinib, with certain mutations conferring intrinsic crizotinib resistance. To overcome this clinical obstacle, our goal was to identify inhibitors with improved potency that can target intractable ALK variants such as F1174L. We find that PF-06463922 has high potency across ALK variants and inhibits ALK more effectively than crizotinib in vitro. Most importantly, PF-06463922 induces complete tumor regression in both crizotinib-resistant and crizotinib-sensitive xenograft mouse models of neuroblastoma, as well as in patient-derived xenografts harboring the crizotinib-resistant F1174L or F1245C mutations. These studies demonstrate that PF-06463922 has the potential to overcome crizotinib resistance and exerts unprecedented activity as a single targeted agent against F1174L and F1245C ALK-mutated xenograft tumors, while also inducing responses in an R1275Q xenograft model. Taken together, these results provide the rationale to move PF-06463922 into clinical trials for treatment of patients with ALK-mutated neuroblastoma. SIGNIFICANCE:The next-generation ALK/ROS1 inhibitor PF-06463922 exerts unparalleled activity in ALK-driven neuroblastoma models with primary crizotinib resistance. Our biochemical and in vivo data provide the preclinical rationale for fast-tracking the development of this agent in children with relapsed/refractory ALK-mutant neuroblastoma.
Project description:The first-in-class inhibitor of ALK, c-MET and ROS1, crizotinib (Xalkori), has shown remarkable clinical efficacy in treatment of ALK-positive non-small cell lung cancer. However, in neuroblastoma, activating mutations in the ALK kinase domain are typically refractory to crizotinib treatment, highlighting the need for more potent inhibitors. The next-generation ALK inhibitor PF-06463922 is predicted to exhibit increased affinity for ALK mutants prevalent in neuroblastoma. We examined PF-06463922 activity in ALK-driven neuroblastoma models in vitro and in vivo In vitro kinase assays and cell-based experiments examining ALK mutations of increasing potency show that PF-06463922 is an effective inhibitor of ALK with greater activity towards ALK neuroblastoma mutants. In contrast to crizotinib, single agent administration of PF-06463922 caused dramatic tumor inhibition in both subcutaneous and orthotopic xenografts as well as a mouse model of high-risk neuroblastoma driven by Th-ALK(F1174L)/MYCN Taken together, our results suggest PF-06463922 is a potent inhibitor of crizotinib-resistant ALK mutations, and highlights an important new treatment option for neuroblastoma patients.
Project description:Activating mutations in the anaplastic lymphoma kinase (ALK) gene were recently discovered in neuroblastoma, a cancer of the developing autonomic nervous system that is the most commonly diagnosed malignancy in the first year of life. The most frequent ALK mutations in neuroblastoma cause amino acid substitutions (F1174L and R1275Q) in the intracellular tyrosine kinase domain of the intact ALK receptor. Identification of ALK as an oncogenic driver in neuroblastoma suggests that crizotinib (PF-02341066), a dual-specific inhibitor of the ALK and Met tyrosine kinases, will be useful in treating this malignancy. Here, we assessed the ability of crizotinib to inhibit proliferation of neuroblastoma cell lines and xenografts expressing mutated or wild-type ALK. Crizotinib inhibited proliferation of cell lines expressing either R1275Q-mutated ALK or amplified wild-type ALK. In contrast, cell lines harboring F1174L-mutated ALK were relatively resistant to crizotinib. Biochemical analyses revealed that this reduced susceptibility of F1174L-mutated ALK to crizotinib inhibition resulted from an increased adenosine triphosphate-binding affinity (as also seen in acquired resistance to epidermal growth factor receptor inhibitors). Thus, this effect should be surmountable with higher doses of crizotinib and/or with higher-affinity inhibitors.
Project description:Oncogenic c-ros oncogene1 (ROS1) fusion kinases have been identified in a variety of human cancers and are attractive targets for cancer therapy. The MET/ALK/ROS1 inhibitor crizotinib (Xalkori, PF-02341066) has demonstrated promising clinical activity in ROS1 fusion-positive non-small cell lung cancer. However, emerging clinical evidence has shown that patients can develop resistance by acquiring secondary point mutations in ROS1 kinase. In this study we characterized the ROS1 activity of PF-06463922, a novel, orally available, CNS-penetrant, ATP-competitive small-molecule inhibitor of ALK/ROS1. In vitro, PF-06463922 exhibited subnanomolar cellular potency against oncogenic ROS1 fusions and inhibited the crizotinib-refractory ROS1(G2032R) mutation and the ROS1(G2026M) gatekeeper mutation. Compared with crizotinib and the second-generation ALK/ROS1 inhibitors ceritinib and alectinib, PF-06463922 showed significantly improved inhibitory activity against ROS1 kinase. A crystal structure of the PF-06463922-ROS1 kinase complex revealed favorable interactions contributing to the high-affinity binding. In vivo, PF-06463922 showed marked antitumor activity in tumor models expressing FIG-ROS1, CD74-ROS1, and the CD74-ROS1(G2032R) mutation. Furthermore, PF-06463922 demonstrated antitumor activity in a genetically engineered mouse model of FIG-ROS1 glioblastoma. Taken together, our results indicate that PF-06463922 has potential for treating ROS1 fusion-positive cancers, including those requiring agents with CNS-penetrating properties, as well as for overcoming crizotinib resistance driven by ROS1 mutation.
Project description:ALK is involved in the onset of several tumors. Crizotinib (XalkoriTM), a potent ALK inhibitor, represents the current front-line treatment for ALK+ NSCLC and shows great clinical efficacy. However, resistant disease often develops after initial response. ASP3026 is a novel second-generation ALK inhibitor with activity on crizotinib-resistant ALK-L1196M gatekeeper mutant. As resistance is likely to be a relevant hurdle for any drug, we sought to determine the resistance profile of ASP3026 in the context of NPM/ALK+ ALCL. We selected six ASP3026-resistant cell lines by culturing human ALCL cells in the presence of increasing concentrations of drug. The established resistant cell lines carry several point mutations in the ALK kinase domain (G1128S, C1156F, I1171N/T, F1174I, N1178H, E1210K and C1156F/D1203N were the most frequent) that are shown to confer resistance to ASP3026 in the Ba/F3 cell model. All mutants were profiled for cross-resistance against a panel of clinically relevant inhibitors including ceritinib, alectinib, crizotinib, AP26113 and PF-06463922. Finally, a genetically heterogeneous ASP3026-resistant cell line was exposed to second-line treatment simulations with all inhibitors. The population evolved according to relative sensitivity of its mutant subclones to the various drugs. Compound PF-06463922 did not allow the outgrowth of any resistant clone, at non-toxic doses.
Project description:Since the original descriptions of gain-of function mutations in anaplastic lymphoma kinase (ALK), interest in the role of this receptor tyrosine kinase in neuroblastoma development and as a potential therapeutic target has escalated. As a group, the activating point mutations in full-length ALK, found in approximately 8% of all neuroblastoma tumors, are distributed evenly across different clinical stages. However, the most frequent somatic mutation, F1174L, is associated with amplification of the MYCN oncogene. This combination of features appears to confer a worse prognosis than MYCN amplification alone, suggesting a cooperative effect on neuroblastoma formation by these two proteins. Indeed, F1174L has shown more potent transforming activity in vivo than the second most common activating mutation, R1275Q, and is responsible for innate and acquired resistance to crizotinib, a clinically relevant ALK inhibitor that will soon be commercially available. These advances cast ALK as a bona fide oncoprotein in neuroblastoma and emphasize the need to understand ALK-mediated signaling in this tumor. This review addresses many of the current issues surrounding the role of ALK in normal development and neuroblastoma pathogenesis, and discusses the prospects for clinically effective targeted treatments based on ALK inhibition.
Project description:We report the preclinical evaluation of PF-06463922, a potent and brain-penetrant ALK/ROS1 inhibitor. Compared with other clinically available ALK inhibitors, PF-06463922 displayed superior potency against all known clinically acquired ALK mutations, including the highly resistant G1202R mutant. Furthermore, PF-06463922 treatment led to regression of EML4-ALK-driven brain metastases, leading to prolonged mouse survival, in a superior manner. Finally, PF-06463922 demonstrated high selectivity and safety margins in a variety of preclinical studies. These results suggest that PF-06463922 will be highly effective for the treatment of patients with ALK-driven lung cancers, including those who relapsed on clinically available ALK inhibitors because of secondary ALK kinase domain mutations and/or brain metastases.
Project description:The anaplastic lymphoma kinase (ALK) gene is overexpressed, mutated or amplified in most neuroblastoma (NB), a pediatric neural crest-derived embryonal tumor. The two most frequent mutations, ALK-F1174L and ALK-R1275Q, contribute to NB tumorigenesis in mouse models, and cooperate with MYCN in the oncogenic process. However, the precise role of activating ALK mutations or ALK-wt overexpression in NB tumor initiation needs further clarification. Human ALK-wt, ALK-F1174L, or ALK-R1275Q were stably expressed in murine neural crest progenitor cells (NCPC), MONC-1 or JoMa1, immortalized with v-Myc or Tamoxifen-inducible Myc-ERT, respectively. While orthotopic implantations of MONC- 1 parental cells in nude mice generated various tumor types, such as NB, osteo/ chondrosarcoma, and undifferentiated tumors, due to v-Myc oncogenic activity, MONC-1-ALK-F1174L cells only produced undifferentiated tumors. Furthermore, our data represent the first demonstration of ALK-wt transforming capacity, as ALK-wt expression in JoMa1 cells, likewise ALK-F1174L, or ALK-R1275Q, in absence of exogenous Myc-ERT activity, was sufficient to induce the formation of aggressive and undifferentiated neural crest cell-derived tumors, but not to drive NB development. Interestingly, JoMa1-ALK tumors and their derived cell lines upregulated Myc endogenous expression, resulting from ALK activation, and both ALK and Myc activities were necessary to confer tumorigenic properties on tumor-derived JoMa1 cells in vitro.
Project description:The ALK kinase inhibitor crizotinib (PF-02341066) is clinically effective in patients with ALK-translocated cancers, but its efficacy will ultimately be limited by acquired drug resistance. Here we report the identification of a secondary mutation in ALK, F1174L, as one cause of crizotinib resistance in a patient with an inflammatory myofibroblastic tumor (IMT) harboring a RANBP2-ALK translocation who progressed while on crizotinib therapy. When present in cis with an ALK translocation, this mutation (also detected in neuroblastomas) causes an increase in ALK phosphorylation, cell growth, and downstream signaling. Furthermore, the F1174L mutation inhibits crizotinib-mediated downregulation of ALK signaling and blocks apoptosis in RANBP2-ALK Ba/F3 cells. A chemically distinct ALK inhibitor, TAE684, and the HSP90 inhibitor 17-AAG are both effective in models harboring the F1174L ALK mutation. Our findings highlight the importance of studying drug resistance mechanisms in order to develop effective clinical treatments for patients with ALK-translocated cancers.
Project description:The ALK(F1174L) mutation is associated with intrinsic and acquired resistance to crizotinib and cosegregates with MYCN in neuroblastoma. In this study, we generated a mouse model overexpressing ALK(F1174L) in the neural crest. Compared to ALK(F1174L) and MYCN alone, co-expression of these two oncogenes led to the development of neuroblastomas with earlier onset, higher penetrance, and enhanced lethality. ALK(F1174L)/MYCN tumors exhibited increased MYCN dosage due to ALK(F1174L)-induced activation of the PI3K/AKT/mTOR and MAPK pathways, coupled with suppression of MYCN pro-apoptotic effects. Combined treatment with the ATP-competitive mTOR inhibitor Torin2 overcame the resistance of ALK(F1174L)/MYCN tumors to crizotinib. Our findings demonstrate a pathogenic role for ALK(F1174L) in neuroblastomas overexpressing MYCN and suggest a strategy for improving targeted therapy for ALK-positive neuroblastoma.
Project description:ALK receptor tyrosine kinase has been shown to be a therapeutic target in neuroblastoma. Germline ALK activating mutations are responsible for the majority of hereditary neuroblastoma and somatic ALK activating mutations are also frequently observed in sporadic cases of advanced NB. Crizotinib, a first-line therapy in the treatment of advanced non-small cell lung cancer (NSCLC) harboring ALK rearrangements, demonstrates striking efficacy against ALK-rearranged NB. However, crizotinib fails to effectively inhibit the activity of ALK when activating mutations are present within its kinase domain, as with the F1174L mutation. Here we show that a new ALK inhibitor AZD3463 effectively suppressed the proliferation of NB cell lines with wild type ALK (WT) as well as ALK activating mutations (F1174L and D1091N) by blocking the ALK-mediated PI3K/AKT/mTOR pathway and ultimately induced apoptosis and autophagy. In addition, AZD3463 enhanced the cytotoxic effects of doxorubicin on NB cells. AZD3463 also exhibited significant therapeutic efficacy on the growth of the NB tumors with WT and F1174L activating mutation ALK in orthotopic xenograft mouse models. These results indicate that AZD3463 is a promising therapeutic agent in the treatment of NB.