F1174L Mutation Research Articles

Identification of different ALK mutations in a pair of neuroblastoma cell lines established at diagnosis and relapse.

Anaplastic Lymphoma Kinase (ALK) is a transmembrane receptor kinase that belongs to the insulin receptor superfamily and has previously been shown to play a role in cell proliferation, migration and invasion in neuroblastoma. Activating ALK mutations are reported in both hereditary and sporadic neuroblastoma tumours, and several ALK inhibitors are currently under clinical evaluation as novel treatments for neuroblastoma. Overall, mutations at codons F1174, R1275 and F1245 together account for ∼85% of reported ALK mutations in neuroblastoma. NBLW and NBLW-R are paired cell lines originally derived from an infant with metastatic MYCN amplified Stage IVS (Evans Criteria) neuroblastoma, at diagnosis and relapse, respectively. Using both Sanger and targeted deep sequencing, this study describes the identification of distinct ALK mutations in these paired cell lines, including the rare R1275L mutation, which has not previously been reported in a neuroblastoma cell line. Analysis of the sensitivity of NBLW and NBLW-R cells to a panel of ALK inhibitors (TAE-684, Crizotinib, Alectinib and Lorlatinib) revealed differences between the paired cell lines, and overall NBLW-R cells with the F1174L mutation were more resistant to ALK inhibitor induced apoptosis compared with NBLW cells. This pair of cell lines represents a valuable pre-clinical model of clonal evolution of ALK mutations associated with neuroblastoma progression.

Novel ALK inhibitor AZD3463 inhibits neuroblastoma growth by overcoming crizotinib resistance and inducing apoptosis.

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.

The ALK/ROS1 Inhibitor PF-06463922 Overcomes Primary Resistance to Crizotinib in ALK-Driven Neuroblastoma.

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. 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.

Abstract 1619: The next-generation ALK inhibitor PF-06463922 overcomes primary resistance to crizotinib in neuroblastoma

Abstract Neuroblastomas (NBs) harboring activating point mutations in the kinase domain of anaplastic lymphoma kinase (ALK) are differentially sensitive to ALK inhibition by crizotinib (Bresler et al. 2011). The F1174L and F1245C amino acid substitutions, which comprise 38.5% (mycancergenome.org) of ALK aberrations in NB, also confer intrinsic resistance to crizotinib and present a significant obstacle in the clinic. Therefore, our goal has been to identify a next-generation ALK inhibitor with improved selectivity and potency to target these resistant mutants effectively. We demonstrate here that PF-06463922 (Pfizer), an ATP-competitive, macrocyclic ALK/ROS1 inhibitor, overcomes crizotinib resistance of these ALK variants in NB, and exerts unprecedented activity as a single agent against the F1174L and F1245C ALK-mutated tumors. At a tenfold lower dose (10 mg/kg) than typically used for crizotinib (100 mg/kg), PF-06463922 induces complete tumor regression in patient-derived (COG-N-426x, F1245C ALK-mutated) and cell line-derived (SY5Y, F1174L ALK-mutated) xenograft mouse models. Whereas crizotinib-treated mice in both models experience rapid tumor growth on therapy, PF-06463922-treated mice showed sustained complete responses for several weeks even after treatment was discontinued. Cell-based and biochemical analyses indicate that the superior activity of PF-06463922 is a function of both higher potency and a broader spectrum of effectiveness across mutants. ATP levels were measured in ten NB cell lines upon treatment with PF-06463922 and crizotinib; at 120 hours post-treatment, PF-06463922 is 26-38 times more effective at inducing growth inhibition in cells with primary resistance than crizotinib. These in vitro and in vivo results are corroborated by our biochemical experiments. In comparative kinase inhibition studies of several next-generation ALK inhibitors, PF-06463922 was most effective against frequently observed ALK variants accounting for approximately 75% of neuroblastoma samples, including those with F1174L mutations. In vitro, PF-06463922 showed at least 3-4 fold higher potency than crizotinib in peptide phosphorylation assays, and its superior inhibition was consistently observed in cellular analysis of transforming ability. Further investigation is necessary to elucidate PF-06463922's mechanism of action and reveal how it induces apoptotic signaling networks without abrogating phospho-ALK. Taken together, these results support the argument that a modified ALK inhibitor may improve outcomes for patients with primary resistance to crizotinib and provide the preclinical basis from which to design a clinical trial for PF-06463922 for the treatment of ALK-mutated NBs. Citation Format: Nicole Infarinato, Jin Park, Renata Sano, Kateryna Krytska, Hannah Ryles, Helen Zou, Nathan Lee, Tod Smeal, Mark A. Lemmon, Yael P. Mosse. The next-generation ALK inhibitor PF-06463922 overcomes primary resistance to crizotinib in neuroblastoma. [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 1619. doi:10.1158/1538-7445.AM2015-1619

SY14-4 - Acquired Resistance to ALK Tyrosine Kinase Inhibitors

ABSTRACT The ALK tyrosine kinase inhibitor crizotinib has demonstrated clinical efficacy in ALK-rearranged NSCLC patients and is currently under additional phase III clinical development as both initial and second-line therapy for advanced ALK-rearranged NSCLC. However, their efficacy will ultimately be limited by the development of acquired drug resistance. Recent studies have identified and studied two mechanisms of ALK TKI resistance based on evidence from a crizotinib-treated ALK-positive lung cancer patient and in a cell line generated from the resistant tumor. To date four secondary mutations, all identified from crizotinib-treated ALK-rearranged tumor patients, have been reported. These mutations either involve the ‘gatekeeper’ residue (L1196) or sites away from crizotinib binding (F1174L, L1152R and C1156Y). The mechanistic basis for how the different mutations lead to crizotinib resistance is not fully understood. The L1196 mutation may create a steric hindrance for crizotinib binding, whereas the F1174L mutation likely promotes the active conformation of ALK, thus disfavoring crizotinib binding which preferentially binds the inactive conformation of ALK. The other mechanism was activation of EGFR signaling as a bypass signaling mechanisms that contributes to ALK inhibitor resistance. Finally, we discuss the strategies that are underway to clinically overcome acquired drug resistance.

The R1275Q Neuroblastoma Mutant and Certain ATP-competitive Inhibitors Stabilize Alternative Activation Loop Conformations of Anaplastic Lymphoma Kinase

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase that, when genetically altered by mutation, amplification, chromosomal translocation or inversion, has been shown to play an oncogenic role in certain cancers. Small molecule inhibitors targeting the kinase activity of ALK have proven to be effective therapies in certain ALK-driven malignancies and one such inhibitor, crizotinib, is now approved for the treatment of EML4-ALK-driven, non-small cell lung cancer. In neuroblastoma, activating point mutations in the ALK kinase domain can drive disease progression, with the two most common mutations being F1174L and R1275Q. We report here crystal structures of the ALK kinase domain containing the F1174L and R1275Q mutations. Also included are crystal structures of ALK in complex with novel small molecule ALK inhibitors, including a classic type II inhibitor, that stabilize previously unobserved conformations of the ALK activation loop. Collectively, these structures illustrate a different series of activation loop conformations than has been observed in previous ALK crystal structures and provide insight into the activating nature of the R1275Q mutation. The novel active site topologies presented here may also aid the structure-based drug design of a new generation of ALK inhibitors.

Abstract 1683: ALK F1174L kinase activity as driver of cell proliferation in neuroblastoma cell line models and neuroblastoma tumors

Abstract Background. Whereas ALK translocations play a role in tumor growth in selected NSCLC cases, mutated forms of ALK have been shown to drive development of neuroblastoma, the most common malignant extracranial tumor of childhood. The mutation F1147L in the oncogene ALK increases the kinase activity, causing deregulated proliferation of sympatho-adrenal progenitor cells. We explored the use of kinase activity measurements to identify ALK activity in cell lysates and patient derived tissue lysates with the aim of identifying biomarkers to trace the activity of ALK in tumors and the effect of ALK inhibitors on these marker signals. Methods. Protein kinase activities were measured on PamChip® peptide microarrays comprising 144 Tyr containing peptide sequences from known human phosphorylation sites. The activity profiling experiments involved recombinant ALK kinase, lysates of cell lines overexpressing ALK and neuroblastoma tumors with elevated ALK F1174L presence. Per microarray analysis 5 ug of total protein was used in the presence of 100 uM ATP. Experiments were performed in the presence and absence of ALK kinase inhibitors crizotinib and NVP-TAE684. Real time kinetics of 144 peptide phosphorylations were measured with a fluorescently labeled antibody. Results. Phosphorylation profiles for neuroblastoma tumors, ALK overexpressing cell lines and recALK were generated. For recALK 48 novel peptide substrates were identified that showed signals depending on both ATP and kinase concentration and inhibition by the ALK inhibitors. In ALK overexpressing cell lines, but not in control cell lysates, signals on many ALK substrate peptides were significantly elevated, and returned to baseline level in the presence of ALK inhibitors. Whereas neuroblastomas with low ALK expression gave similar kinase activity profiles, the tumors harboring the ALK F1174L mutation were different, suggesting a more complex regulation of phosphorylation activity in these tumors. All tumors showed on-chip responses with ALK inhibitor drugs, but to different extent, which could be a basis for response prediction biomarker discovery. Conclusion. ALK activity of recombinant kinases, cell lysates and tumor lysates can be detected on PamChip® peptide microarrays. Based on results with model systems, this method shows promise to identify tumors that express elevated ALK activity and to study response of this class to ALK inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1683. doi:1538-7445.AM2012-1683

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Abstract 1528: Cooperative cross-talk between neuroblastoma subtypes confers resistance to anaplastic lymphoma kinase inhibition

Abstract Neuroblastoma is a pediatric solid tumor that can be stratified into stroma-rich and stroma-poor histological subgroups. The stromal compartment of neuroblastoma is composed mostly of Schwann cells and they play critical roles in the differentiation, survival and angiogenic responses of tumor cells. In certain neuroblastoma cell lines, the coexistence of neuroblastic N-type and substrate-adherent S-type is frequently observed. One such cell line, SK-N-SH, harbors a F1174L oncogenic mutation in the Anaplastic Lymphoma Kinase (ALK) gene. Treatment of SK-N-SH with an ALK chemical inhibitor, TAE684, resulted in the outgrowth of S-type cells that expressed the Schwann cell marker, S100α6. Nucleotide sequencing analysis of these TAE684-resistant (TR) sublines revealed the presence of the ALK F1174L mutation suggesting their tumor origin, although ALK protein was not detected. Consistent with these findings, TR cells displayed ∼9-fold higher IC50 values than N-type cells. Also, unlike N-type cells, TR cells have readily detectable phosphorylated STAT3 but weaker phosphorylated AKT. Under co-culture conditions, TR cells conferred survival to N-type cells against the apoptotic effect of TAE684. Co-cultivation also greatly enhanced the overall phosphorylation of STAT3 and its transcriptional activity in N-type cells. Finally, conditioned medium from TR clones enhanced cell viability of N-type cells and this effect was Phosphatidylinositol 3-Kinase-dependent. Taken together, these results demonstrate the ability of tumor-derived S-type cells in protecting N-type cells against the apoptotic effect of an ALK kinase inhibitor through upregulating pro-survival signaling. This project was supported by MACC funds, Advancing a Healthier Wisconsin, Wisconsin Breast Cancer Showhouse (O.W.), NIH CA133669 (H.G. and A.M.C.) and the Children's Research Institutes of the Children's Hospital of Wisconsin. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1528. doi:1538-7445.AM2012-1528

Internalization and Down-Regulation of the ALK Receptor in Neuroblastoma Cell Lines upon Monoclonal Antibodies Treatment

Recently, activating mutations of the full length ALK receptor, with two hot spots at positions F1174 and R1275, have been characterized in sporadic cases of neuroblastoma. Here, we report similar basal patterns of ALK phosphorylation between the neuroblastoma IMR-32 cell line, which expresses only the wild-type receptor (ALKWT), and the SH-SY5Y cell line, which exhibits a heterozygous ALK F1174L mutation and expresses both ALKWT and ALKF1174L receptors. We demonstrate that this lack of detectable increased phosphorylation in SH-SY5Y cells is a result of intracellular retention and proteasomal degradation of the mutated receptor. As a consequence, in SH-SY5Y cells, plasma membrane appears strongly enriched for ALKWT whereas both ALKWT and ALKF1174L were present in intracellular compartments. We further explored ALK receptor trafficking by investigating the effect of agonist and antagonist mAb (monoclonal antibodies) on ALK internalization and down-regulation, either in SH-SY5Y cells or in cells expressing only ALKWT. We observe that treatment with agonist mAbs resulted in ALK internalization and lysosomal targeting for receptor degradation. In contrast, antagonist mAb induced ALK internalization and recycling to the plasma membrane. Importantly, we correlate this differential trafficking of ALK in response to mAb with the recruitment of the ubiquitin ligase Cbl and ALK ubiquitylation only after agonist stimulation. This study provides novel insights into the mechanisms regulating ALK trafficking and degradation, showing that various ALK receptor pools are regulated by proteasome or lysosome pathways according to their intracellular localization.

The right type of ALK

The anaplastic lymphoma kinase (ALK) was recently found to be mutated in neuroblastoma. Scott Bresler and colleagues used ALK-expressing neuroblastoma cell lines and xenografts to assess the efficacy of the ALK and MET tyrosine kinase inhibitor crizotinib. They found that neuroblastoma cell lines that had either amplified wild-type ALK or the ALK mutant R1275Q were sensitive to crizotinib. However, neuroblastoma cell lines with F1174L ALK mutations were relatively resistant to this drug, owing to the increased ATP-binding affinity of this mutant. The authors propose that patients with neuroblastomas that have the F1174L mutation might still respond to either high doses of crizotinib or to ALK inhibitors that have an increased binding affinity compared with crizotinib.

ALK Mutants Show Differential Response to Crizotinib

Abstract Major finding: F1174L-mutated ALK shows reduced sensitivity to crizotinib inhibition. Mechanism: The F1174L mutation enhances the ATP-binding affinity of ALK. Impact: Patients with F1174L-mutated ALK may benefit from higher crizotinib doses.

Crizotinib‐Resistant Mutants of EML4‐ALK Identified Through an Accelerated Mutagenesis Screen

Activating gene rearrangements of anaplastic lymphoma kinase (ALK) have been identified as driver mutations in non-small-cell lung cancer, inflammatory myofibroblastic tumors, and other cancers. Crizotinib, a dual MET/ALK inhibitor, has demonstrated promising clinical activity in patients with non-small-cell lung cancer and inflammatory myofibroblastic tumors harboring ALK translocations. Inhibitors of driver kinases often elicit kinase domain mutations that confer resistance, and such mutations have been successfully predicted using in vitro mutagenesis screens. Here, this approach was used to discover an extensive set of ALK mutations that can confer resistance to crizotinib. Mutations at 16 residues were identified, structurally clustered into five regions around the kinase active site, which conferred varying degrees of resistance. The screen successfully predicted the L1196M, C1156Y, and F1174L mutations, recently identified in crizotinib-resistant patients. In separate studies, we demonstrated that crizotinib has relatively modest potency in ALK-positive non-small-cell lung cancer cell lines. A more potent ALK inhibitor, TAE684, maintained substantial activity against mutations that conferred resistance to crizotinib. Our study identifies multiple novel mutations in ALK that may confer clinical resistance to crizotinib, suggests that crizotinib's narrow selectivity window may underlie its susceptibility to such resistance and demonstrates that a more potent ALK inhibitor may be effective at overcoming resistance.

Cooperative Cross-Talk between Neuroblastoma Subtypes Confers Resistance to Anaplastic Lymphoma Kinase Inhibition

Neuroblastoma is a pediatric solid tumor that can be stratified into stroma-rich and stroma-poor histological subgroups. The stromal compartment of neuroblastoma is composed mostly of Schwann cells, and they play critical roles in the differentiation, survival, and angiogenic responses of tumor cells. In certain neuroblastoma cell lines, the coexistence of neuroblastic N-type and substrate-adherent S-type is frequently observed. One such cell line, SK-N-SH, harbors a F1174L oncogenic mutation in the anaplastic lymphoma kinase (ALK) gene. Treatment of SK-N-SH with an ALK chemical inhibitor, TAE684, resulted in the outgrowth of S-type cells that expressed the Schwann cell marker, S100α6. Nucleotide sequencing analysis of these TAE684-resistant (TR) sublines revealed the presence of the ALK F1174L mutation, suggesting their tumor origin, although ALK protein was not detected. Consistent with these findings, TR cells displayed approximately 9-fold higher IC(50) values than N-type cells. Also, unlike N-type cells, TR cells have readily detectable phosphorylated STAT3 but weaker phosphorylated AKT. Under coculture conditions, TR cells conferred survival to N-type cells against the apoptotic effect of TAE684. Cocultivation also greatly enhanced the overall phosphorylation of STAT3 and its transcriptional activity in N-type cells. Finally, conditioned medium from TR clones enhanced cell viability of N-type cells, and this effect was phosphatidylinositol 3-kinase dependent. Taken together, these results demonstrate the ability of tumor-derived S-type cells in protecting N-type cells against the apoptotic effect of an ALK kinase inhibitor through upregulating prosurvival signaling.

Abstract 4685: Mechanistic guidance of ALK inhibition for the treatment of neuroblastoma

Abstract Receptor tyrosine kinases (RTKs) are important therapeutic targets in a wide variety of human diseases, especially neoplastic disorders. Several drugs that target the tyrosine kinase domains of the epidermal growth factor receptor (EGFR) or other tyrosine kinases are now in clinical use, and it has become clear that their use requires guidance from the results of genetic, biochemical and structural studies of their targets. A similar situation is emerging with another RTK-the anaplastic lymphoma kinase (ALK)-in neuroblastoma (NB), where activating mutations are therapeutic targets. In our study, inhibition of in vitro proliferation of NB cell lines was associated with abrogation of phosphorylation of ALK by PF-02341066, an orally bioavailable, ATP-competitive inhibitor of ALK and c-Met. Cell lines expressing F1174L were observed to be resistant to PF-02341066 compared to cells expressing R1275Q in vitro. Additionally, the inhibitor caused complete and sustained regression of xenografts harboring the R1275Q mutation which accounts for ∼50% of all mutations in human NB. However, the compound displayed modest tumor growth delay against xenografts harboring F1174L mutations. Little is known about the normal mechanism of activation of this kinase, and our data indicate that, under the proper conditions, classical trans-autophosphorylation of the activation loop in the recombinantly expressed tyrosine kinase domain (TKD) results in a marked (∼80 fold) increase in catalytic efficiency. Recent crystal structures of ALK-TKD have revealed, surprisingly, that autoinhibition is achieved by a mechanism similar to that of EGFR rather than other insulin receptor family members. However, ALK relieves this autoinhibition solely by autophosphorylation whereas EGFR depends on allosteric interactions between dimerized domains. Our kinetic data indicate that the R1275Q and F1174L mutations perturb autoinhibitory features of this kinase and activate the TKD to varying degrees, with the F1174L mutant displaying more dramatic dysregulation. Furthermore, F1174L displayed a lower Km, ATP value compared to R1275Q, directly analogous to the EGFR L858R/T790M resistant double mutant compared to EGFR harboring the L858R mutation alone. Additionally, F1174L was nearly identical to wild-type and R1275Q ALK in screens using a panel of commercially available inhibitors. This suggests that the reason for the relative resistance of F1174L-expressing cell lines to PF-02341066 is due primarily to stronger ATP affinity. This study provides preclinical rationale for the use of PF-02341066 in NB as well as important insight into how ALK is regulated in normal and disease states. To effectively target F1174L, an innate resistance mechanism for NB cells expressing it, as well as an acquired resistance mechanism for ALK-translocated tumors, we propose that next-generation ALK inhibitors should bind the ATP-pocket with higher affinity or irreversibly. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4685. doi:10.1158/1538-7445.AM2011-4685

Abstract 1541: Secondary mutations in ALK and resistance to ALK kinase inhibitors

Abstract Background: The anaplastic lymphoma kinase (ALK) inhibitor crizotinib is clinically effective in cancer patients whose tumors harbor ALK translocations. However, drug resistance will ultimately develop in most if not all patients. We studied drug resistance mechanisms using both in vitro models and analyses from tumors of patients that had developed crizotinib resistance. Methods and Results: We identified a secondary mutation in ALK, F1174L, as one cause of crizotinib resistance in a patient with an inflammatory myofibroblastic tumor (IMT) harbouring a RANBP2-ALK translocation who progressed while 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. We also evaluated the impact of mutations in the ALK gatekeeper residue (L1196) and engineered all possible gatekeeper mutations resulting from a single nucleotide change into the background of EML4-ALK. Three of the mutations (L1196M, L1196Q and L1196V) in the background of EML4-ALK led to IL-3 independent growth. Furthermore the EML4-ALK L1196M mutant resulted in a greater baseline ALK phosphorylation, cell growth and downstream signaling compared to EML4-ALK alone. A chemically distinct ALK inhibitor, TAE684, or the HSP90 inhibitor 17-AAG are both effective in models harbouring the F1174L or the L1196M ALK mutations. Conclusions: We identify secondary mutations in ALK that confer crizotinib resistance. In addition, we identify therapeutic strategies, alternative ALK inhibitors or HSP90 inhibitors that may be clinically effective in patients that have developed crizotinib resistance. Understanding the mechanistic basis of drug resistance is necessary in order to develop effective clinical treatments for patients with ALK-translocated cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1541. doi:10.1158/1538-7445.AM2011-1541

The neuroblastoma-associated F1174L ALK mutation causes resistance to an ALK kinase inhibitor in ALK-translocated cancers.

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.

Abstract 5248: Neuroblastoma tumors with different ALK mutations are sensitive to Perifosine

Abstract Background: Neuroblastoma (NB) is the most common extracranial solid tumor of children. Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase predominantly expressed in the developing nervous system. Recently, mutated ALK has been identified in association with familial and sporadic neuroblastoma (NB). Patients whose tumors contain amplification, mutation or express increased levels of activated ALK have a poor prognosis. Downstream targets of ALK include the STAT and Akt pathways. Perifoisine is an Akt inhibitor that has been reported to inhibit tumor growth in adult pre-clinical models. The aim of this study was to assess whether ALK mutations affect the response of NB cells to perifosine. Methods: We selected 4 NB cell lines with different ALK mutation status (SY5Y-ALK F1174L mutation; KCNR-ALK R1275Q mutation, AS and NGP with wild-type (wt) ALK. The effect of perifosine on Akt phosphorylation and cell survival was evaluated in these cell lines. In vivo studies were also performed in nude mice by subcutaneous injection of NB cells with or without ALK mutation. The tumor growth and phosphorylated Akt levels in tumor tissues were studied. Results: In vitro, treatment with perifosine induced decreases in Akt phosphorylation in all the 4 cell lines. SY5Y-ALK F1174L which is reported to be sensitive to ALK inhibitor TAE684, and KCNR-ALK R1275Q, AS-ALK wt and NGP-ALK wt which are reported to be resistant to ALK inhibitor TAE684, all responded to perifosine treatment. In vivo, perifosine treatment delayed or inhibited growth of tumors containing mutant or wild-type ALK. The phosphorylated Akt levels were lower in tumors treated with perifosine compared to tumors treated with control reagent. Conclusions: These data indicate that Perifosine is able to control NB tumor cell growth despite the mutational status of ALK. Perifosine may be a candidate treatment for NB tumors not only for those with wt ALK but especially for those that carry mutations that are resistant to ALK inhibitor treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5248.

Abstract SY14-02: ALK in neuroblastoma

Abstract Neuroblastoma is a cancer of early childhood that arises from the developing autonomic nervous system. It is the most common malignancy diagnosed in the first year of life and shows a wide range of clinical phenotypes with some patients having tumors that regress spontaneously, whereas the majority of patients have aggressive metastatic disease. Neuroblastoma remains an important clinical problem as it continues to be a leading cause of childhood cancer mortality despite dramatic escalations in dose-intensive chemo radiotherapy, and long-term survivors experience significant treatment related morbidity. To improve outcome and make paradigm-shifting advances in this disease, it is necessary to discover the key oncogenic drivers of the malignant process and exploit these therapeutically. We recently discovered activating mutations within the tyrosine kinase domain of the anaplastic lymphoma kinase (ALK) oncogene as the major cause of hereditary neuroblastoma, and showed that somatically acquired mutations and gene amplification events often drive the malignant process. Here we report a comprehensive survey of ALK genomic status in all neuroblastoma phenotypic subsets and perform preclinical validation that targeted inhibition of activated ALK kinase is an effective therapeutic strategy. ALK mutations were detected in 7% of cases, were restricted to the tyrosine kinase domain and occurred equally in all patient subsets. The most common germline mutation in familial cases (R1275Q) was also the most common somatic mutation (49% of tumors with a mutation). High-level amplification of ALK was detected in 2.4% of tumors, regional gain of the ALK locus in an additional 19% of cases, and both were associated with differentially increased ALK mRNA expression (P= 0.004). ALK amplification is strongly associated with the high-risk subset (P<0.001) and MYCN amplification (P<0.001). Future studies will need to focus on correlation of pathway activation status and/or sensitivity to targeted interruption of ALK signaling with underlying genomic status. In addition, in order to perform a proper clinical correlative study to determine the independent prognostic impact of ALK aberration, a large meta-analysis will need to be performed that has a sample size 2-3x larger than the number of cases studied here. This is required because relapse and death is rare in the low- and intermediate-risk group of patients, and because other confounders such as MYCN amplification co-occur. A 1500 subject clinical correlative study is ongoing. A major challenge in the field is to distinguish driver mutations that are responsible for malignant transformation from nonfunctional passenger mutations that may arise in the setting of genomic instability. We provide functional evidence of this concept with experimental validation of four of the most common potential driver mutations, each resulting in constitutive kinase activation of the ALK kinase, but in varying degrees. The two most commonly observed mutations resulted in robust constitutive phosphorylation of the ALK kinase, whereas the rare germline mutation G1128A, which has not been observed somatically, resulted in only weak activation, similar to the amount seen with forced over expression of wild-type protein. Pharmacologic inhibition of ALK kinase activity with PF-02341066, a dual c-Met/ALK inhibitor, demonstrated in vitro cytotoxicity in an ALK genomic status dependant manner. Cytotoxicity correlated with abrogation of constitutively activated ALK phosphorylation. Likewise, oral dosing of PF-02341066 in immunocompromised mice xenotransplanted with human neuroblastoma-derived cell lines resulted in differential anti-tumor activity in a genomic status-dependant manner. Xenografts harboring the R1275Q mutation showing complete regression, while those harboring an F1174L mutation showed significant tumor growth delay (P < 0.0001). Xenografts with no evidence for constitutively phosphorylated ALK showed no anti-tumor activity. Computational homology modeling based upon the crystal structure of PF-02341066 bound to c-Met predicted that F1174L destabilizes the preferred protein conformation for PF-02341066 binding, providing in silico evidence for the mutation-specific differential sensitivity to PF-02341066. Our data clearly demonstrate that cytotoxicity to PF-02341066 is highly associated with ALK genomic status and evidence for constitutive activation. If preclinical models are predictive of success in the ongoing and planned clinical trials, and anti-tumor response is restricted to patients with neuroblastomas that have aberrant ALK signaling, it will be imperative to obtain tumor tissue to identify the patients most likely to benefit from ALK-targeted therapy. Understanding the genetic heterogeneity of neuroblastoma tumors and devising strategies to circumvent their rapid selection for resistance to targeted ALK inhibitors will guide the design of second-generation targeted therapies. In addition, knowledge of resistant mutations should permit the development of assays to detect drug-resistant clones before clinical relapse. Citation Format: Yael P. Mosse. ALK in neuroblastoma [abstract]. In: Proceedings of the AACR 101st Annual Meeting 2010; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr SY14-02

Inhibition of ALK mutated neuroblastomas by the selective inhibitor PF-02341066

10008b Background: Mutations in the ALK oncogene are the major cause of hereditary neuroblastoma (NB), and may offer a tractable therapeutic target. Methods: We resequenced the ALK tyrosine kinase domain in 496 primary NBs. We integrated data from 76 primary tumors analyzed using Illumina HumanHap550 SNP and Human-6 mRNA expression arrays. We stained a tissue microarray (TMA) of 134 NBs with anti-ALK and anti-pALK antibodies. We evaluated the in vitro and in vivo activity of PF-02341066, a small molecule inhibitor of ALK. Computational chemistry was used to homology map ALK interaction with PF-02341066. Results: Mutations were discovered in 38/496 NBs (8%), across the spectrum of phenotypes. R1275Q substitutions were most common (45%), followed by F1174L (15%). Evaluation of matched constitutional DNAs demonstrated 10% carried occult germline mutations. Integration of primary tumor microarrays from 76 samples showed that ALK mRNA expression was highly correlated with ALK DNA copy number (p=0.0005). ALK protein was nearly universally expressed (129/134; 93%), and there was evidence for ALK activation in 15% of tumors (pALK moderate or strong staining in 20/134), suggesting an additional and alternative pathway to ALK activation. A panel of 18 NB cell lines showed differential in vitro sensitivity to PF-2341066 dependent on ALK mutation and copy number status. A wild type (WT) amplified line and 2 lines harboring R1275Q mutations were sensitive (IC50 145 - 301 nM). Lines harboring F1174L mutations were more resistant (IC50 >950nM) as were lines with no ALK aberrations. Inhibition of proliferation was associated with abolishing phosphorylation of ALK, STAT3 and AKT. PF-02341066 caused complete regression of R1275Q xenografts, but caused only minimal growth delay in F1174L and WT xenografts. Based on homology mapping to a crystal structure of PF-02341066 bound to c-Met, the F1174L mutation is predicted to destabilize the specific conformation of the kinase activation loop required for binding PF-02341066. Conclusions: ALK pathway activation occurs in at least 15% of human neuroblastomas. PF-02341066 is a potent inhibitor of the most common ALK mutation, but there is a structural basis for resistance to F1174L mutations. Co-crystallization studies are ongoing and a phase 1 study is planned. No significant financial relationships to disclose.