Will the clinical development of 4th-generation “double mutant active” ALK TKIs (TPX-0131 and NVL-655) change the future treatment paradigm of ALK+ NSCLC?

Our current treatment paradigm of advanced anaplastic lymphoma kinase fusion (ALK+) non-small cell lung cancer (NSCLC) classifies the six currently approved ALK tyrosine kinase inhibitors (TKIs) into three generations. The 2nd-generation (2G) and 3rd-generation (3G) ALK TKIs are all “single mutant active” with varying potencies across a wide spectrum of acquired single ALK resistance mutations. There is a vigorous debate among clinicians which is the best upfront ALK TKI is for the first-line (1L) treatment of ALK+ NSCLC and the subsequent sequencing strategies whether it should be based on the presence of specific on-target ALK resistance mutations or not. Regardless, sequential use of “single mutant active” ALK TKIs will eventually lead to double ALK resistance mutations in cis. This has led to the creation of fourth generation (4G) “double mutant active” ALK TKIs such as TPX-0131 and NVL-655. We discuss the critical properties 4G ALK TKIs must possess to be clinically successful. We proposed conceptual first-line, second-line, and molecularly-based third-line registrational randomized clinical trials designed for these 4G ALK TKIs. How these 4G ALK TKIs would be used in the future will depend on which line of treatment the clinical trial design(s) is adopted provided the trial is positive. If approved, 4G ALK TKIs may usher in a new treatment paradigm for advanced ALK+ NSCLC that is based on classifying ALK TKIs based on the intrinsic functional capabilities (“singe mutant active” versus “double mutant active”) rather than the loosely-defined “generational” (first-, second-,third-,fourth-) classification and avoid the current clinical approaches of seemingly random sequential use of 2G and 3G ALK TKIs.     

Will the clinical development of 4th-generation “double mutant active” ALK TKIs (TPX-0131 and NVL-655) change the future treatment paradigm of ALK+ NSCLC?

Our current treatment paradigm of advanced anaplastic lymphoma kinase fusion (ALK+) non-small cell lung cancer (NSCLC) classifies the six currently approved ALK tyrosine kinase inhibitors (TKIs) into three generations. The 2nd-generation (2G) and 3rd-generation (3G) ALK TKIs are all “single mutant active” with varying potencies across a wide spectrum of acquired single ALK resistance mutations. There is a vigorous debate among clinicians which is the best upfront ALK TKI is for the first-line (1L) treatment of ALK+ NSCLC and the subsequent sequencing strategies whether it should be based on the presence of specific on-target ALK resistance mutations or not. Regardless, sequential use of “single mutant active” ALK TKIs will eventually lead to double ALK resistance mutations in cis. This has led to the creation of fourth generation (4G) “double mutant active” ALK TKIs such as TPX-0131 and NVL-655. We discuss the critical properties 4G ALK TKIs must possess to be clinically successful. We proposed conceptual first-line, second-line, and molecularly-based third-line registrational randomized clinical trials designed for these 4G ALK TKIs. How these 4G ALK TKIs would be used in the future will depend on which line of treatment the clinical trial design(s) is adopted provided the trial is positive. If approved, 4G ALK TKIs may usher in a new treatment paradigm for advanced ALK+ NSCLC that is based on classifying ALK TKIs based on the intrinsic functional capabilities (“singe mutant active” versus “double mutant active”) rather than the loosely-defined “generational” (first-, second-,third-,fourth-) classification and avoid the current clinical approaches of seemingly random sequential use of 2G and 3G ALK TKIs.     

ALK1 signalling analysis identifies angiogenesis related genes and reveals disparity between TGF-β and constitutively active receptor induced gene expression

Background

TGF-β1 is an important angiogenic factor involved in the different aspects of angiogenesis and vessel maintenance. TGF-β signalling is mediated by the TβRII/ALK5 receptor complex activating the Smad2/Smad3 pathway. In endothelial cells TGF-β utilizes a second type I receptor, ALK1, activating the Smad1/Smad5 pathway. Consequently, a perturbance of ALK1, ALK5 or TβRII activity leads to vascular defects. Mutations in ALK1 cause the vascular disorder hereditary hemorrhagic telangiectasia (HHT).

Methods

The identification of ALK1 and not ALK5 regulated genes in endothelial cells, might help to better understand the development of HHT. Therefore, the human microvascular endothelial cell line HMEC-1 was infected with a recombinant constitutively active ALK1 adenovirus, and gene expression was studied by using gene arrays and quantitative real-time PCR analysis.

Results

After 24 hours, 34 genes were identified to be up-regulated by ALK1 signalling. Analysing ALK1 regulated gene expression after 4 hours revealed 13 genes to be up- and 2 to be down-regulated. Several of these genes, including IL-8, ET-1, ID1, HPTPη and TEAD4 are reported to be involved in angiogenesis. Evaluation of ALK1 regulated gene expression in different human endothelial cell types was not in complete agreement. Further on, disparity between constitutively active ALK1 and TGF-β1 induced gene expression in HMEC-1 cells and primary HUVECs was observed.

Conclusion

Gene array analysis identified 49 genes to be regulated by ALK1 signalling and at least 14 genes are reported to be involved in angiogenesis. There was substantial agreement between the gene array and quantitative real-time PCR data. The angiogenesis related genes might be potential HHT modifier genes. In addition, the results suggest endothelial cell type specific ALK1 and TGF-β signalling.     

Pulmonary Sarcomatoid Carcinoma with ALK Rearrangement: Frequency,Clinical-Pathologic Characteristics,and Response to ALK Inhibitor

PURPOSE: The incidence of anaplastic lymphoma kinase (ALK) rearrangement in pulmonary sarcomatoid carcinoma (PSC) is controversial. In this study, we aimed to reveal the reliable frequency and the clinical-pathologic characteristics of pulmonary sarcomatoid carcinoma (PSC) with ALK rearrangement in Chinese population, and to provide insight into the translatability of anti-ALK treatment in this treatment-refractory disease. METHODS: Immunohistochemistry (IHC) using a Ventana anti-ALK (D5F3) rabbit monoclonal antibody was performed in 141 PSC specimens collected from multiple medical centers. IHC-positive cases were then confirmed using ALK fluorescent in situ hybridization (FISH). The incidence rates and clinical-pathologic characteristics of ALK-rearranged PSC were then analyzed. Response to ALK inhibitor crizotinib in a patient with ALK-rearranged PSC was evaluated according to the response evaluation criteria for solid tumors (RECIST) version 1.1. RESULTS: Five of 141 (3.5%) of PSCs showed ALK rearrangement-positive by IHC and then were confirmed by FISH. Two were carcinosarcomas and the other three were pulmonary pleomorphic carcinoma (PPC). Strong positive ALK rearrangement was observed in both the epithelioid and sarcomatoid components. The median age of ALK-positive patients was younger than that of ALK-negative patients. PSCs in never-smokers were more likely to harbor ALK rearrangement than those in former or current smokers (P < .05). A 40-year-old woman diagnosed with ALK-rearranged PPC experienced a partial response (?32%) to the ALK inhibitor crizotinib. CONCLUSIONS: The incidence rates of ALK rearrangement in PSC in the Chinese population are similar to those of other subtypes of NSCLC. PSCs in younger never-smokers are more often to harbor ALK rearrangement. ALK inhibitors may serve as an effective treatment for ALK-rearranged PSC.     

TGFβ/BMP Type I Receptors ALK1 and ALK2 Are Essential for BMP9-induced Osteogenic Signaling in Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are bone marrow stromal cells that can differentiate into multiple lineages. We previously demonstrated that BMP9 is one of the most potent BMPs to induce osteogenic differentiation of MSCs. BMP9 is one of the least studied BMPs. Whereas ALK1, ALK5, and/or endoglin have recently been reported as potential BMP9 type I receptors in endothelial cells, little is known about type I receptor involvement in BMP9-induced osteogenic differentiation in MSCs. Here, we conduct a comprehensive analysis of the functional role of seven type I receptors in BMP9-induced osteogenic signaling in MSCs. We have found that most of the seven type I receptors are expressed in MSCs. However, using dominant-negative mutants for the seven type I receptors, we demonstrate that only ALK1 and ALK2 mutants effectively inhibit BMP9-induced osteogenic differentiation in vitro and ectopic ossification in MSC implantation assays. Protein fragment complementation assays demonstrate that ALK1 and ALK2 directly interact with BMP9. Likewise, RNAi silencing of ALK1 and ALK2 expression inhibits BMP9-induced BMPR-Smad activity and osteogenic differentiation in MSCs both in vitro and in vivo. Therefore, our results strongly suggest that ALK1 and ALK2 may play an important role in mediating BMP9-induced osteogenic differentiation. These findings should further aid us in understanding the molecular mechanism through which BMP9 regulates osteogenic differentiation of MSCs.     

Anaplastic Lymphoma Kinase Gene Copy Number Gain in Inflammatory Breast Cancer (IBC): Prevalence,Clinicopathologic Features and Prognostic Implication

Background

Inflammatory breast cancer (IBC) is the most aggressive form of breast cancer, and its molecular pathogenesis still remains to be elucidated. This study aimed to evaluate the prevalence and implication of anaplastic lymphoma kinase (ALK) copy number change in IBC patients.

Methods

We retrospectively collected formalin-fixed, paraffin-embedded tumor tissues and medical records of IBC patients from several institutes in Korea. ALK gene copy number change and rearrangement were assessed by fluorescence in situ hybridization (FISH) assay, and ALK expression status was evaluated by immunohistochemical (IHC) staining.

Results

Thirty-six IBC patients including those with HER2 (+) breast cancer (16/36, 44.4%) and triple-negative breast cancer (13/36, 36.1%) were enrolled in this study. ALK copy number gain (CNG) was observed in 47.2% (17/36) of patients, including one patient who harbored ALK gene amplification. ALK CNG (+) patients showed significantly worse overall survival compared to ALK CNG (-) patients in univariate analysis (24.9 months vs. 38.1 months, p = 0.033). Recurrence free survival (RFS) after curative mastectomy was also significantly shorter in ALK CNG (+) patients than in ALK CNG (-) patients (n = 22, 12.7 months vs. 43.3 months, p = 0.016). Multivariate Cox regression analysis with adjustment for HER2 and ER statuses showed significantly poorer RFS for ALK CNG (+) patients (HR 5.63, 95% CI 1.11–28.44, p = 0.037).

Conclusion

This study shows a significant presence of ALK CNG in IBC patients, and ALK CNG was associated with significantly poorer RFS.     

Discovery of 3-(4-sulfamoylnaphthyl)pyrazolo[1,5-a]pyrimidines as potent and selective ALK2 inhibitors

The pyrazolo[1,5-a]pyrimidine LDN-193189 is a potent inhibitor of activin receptor-like kinase 2 (ALK2) but is nonselective for highly homologous ALK3 and shows only modest kinome selectivity. Herein, we describe the discovery of a novel series of potent and selective ALK2 inhibitors by replacing the quinolinyl with a 4-(sulfamoyl)naphthyl, yielding ALK2 inhibitors that exhibit not only excellent discrimination versus ALK3 but also high kinome selectivity. In addition, the optimized compound 23 demonstrates good ADME and in vivo pharmacokinetic properties.     

The Molecular Detection and Clinical Significance of ALK Rearrangement in Selected Advanced Non-Small Cell Lung Cancer: ALK Expression Provides Insights into ALK Targeted Therapy

Background

This study aimed to elucidate clinical significance of anaplastic lymphoma kinase (ALK) rearrangement in selected advanced non-small cell lung cancer (NSCLC), to compare the application of different ALK detection methods, and especially evaluate a possible association between ALK expression and clinical outcomes in crizotinib-treated patients.

Methods

ALK status was assessed by fluorescent in situ hybridization (FISH), immunohistochemistry (IHC) and quantitative RT-PCR (qRT-PCR) in 173 selected advanced NSCLC patients. Clinicopathologic data, genotype status and survival outcomes were analyzed. Moreover, the association of ALK expression with clinical outcomes was evaluated in ALK FISH-positive crizotinib-treated patients including two patients with concurrent epidermal growth factor receptor (EGFR) mutation.

Results

The positivity detection rate of ALK rearrangement by FISH, IHC and qRT-PCR was 35.5% (59/166), 35.7% (61/171), and 27.9% (34/122), respectively. ALK rearrangement was observed predominantly in young patients, never or light smokers, and adenocarcinomas, especially with signet ring cell features and poor differentiation. Median progression-free survival (PFS) of crizotinib-treated patients was 7.6 months. The overall survival (OS) of these patients was longer compared with that of crizotinib-naive or wild-type cohorts, but there was no significant difference in OS compared with patients with EGFR mutation. ALK expression did not associate with PFS; but, when ALK expression was analyzed as a dichotomous variable, moderate and strong ALK expression had a decreased risk of death (P = 0.026). The two patients with concomitant EGFR and ALK alterations showed difference in ALK expression, response to EGFR and ALK inhibitors, and overall survival.

Conclusions

Selective enrichment according to clinicopathologic features in NSCLC patients could highly improve the positivity detection rate of ALK rearrangement for ALK-targeted therapy. IHC could provide more clues for clinical trial design and therapeutic strategies for ALK-positive NSCLC patients including patients with double genetic aberration of ALK and EGFR.     

Platform Comparison for Evaluation of ALK Protein Immunohistochemical Expression,Genomic Copy Number and Hotspot Mutation Status in Neuroblastomas

ALK is an established causative oncogenic driver in neuroblastoma, and is likely to emerge as a routine biomarker in neuroblastoma diagnostics. At present, the optimal strategy for clinical diagnostic evaluation of ALK protein, genomic and hotspot mutation status is not well-studied. We evaluated ALK immunohistochemical (IHC) protein expression using three different antibodies (ALK1, 5A4 and D5F3 clones), ALK genomic status using single-color chromogenic in situ hybridization (CISH), and ALK hotspot mutation status using conventional Sanger sequencing and a next-generation sequencing platform (Ion Torrent Personal Genome Machine (IT-PGM)), in archival formalin-fixed, paraffin-embedded neuroblastoma samples. We found a significant difference in IHC results using the three different antibodies, with the highest percentage of positive cases seen on D5F3 immunohistochemistry. Correlation with ALK genomic and hotspot mutational status revealed that the majority of D5F3 ALK-positive cases did not possess either ALK genomic amplification or hotspot mutations. Comparison of sequencing platforms showed a perfect correlation between conventional Sanger and IT-PGM sequencing. Our findings suggest that D5F3 immunohistochemistry, single-color CISH and IT-PGM sequencing are suitable assays for evaluation of ALK status in future neuroblastoma clinical trials.     

Protective Effect of Aliskiren in Experimental Ischemic Stroke: Up-Regulated p-PI3K,p-AKT,Bcl-2 Expression,Attenuated Bax Expression

Aliskiren (ALK), a pharmacological renin inhibitor, is an effective antihypertensive drug and has potent anti-apoptotic activity, but it is currently unknown whether ALK is able to attenuate brain damage caused by acute cerebral ischemia independent of its blood pressure-lowering effects. This study aimed to investigate the role of ALK and its potential mechanism in cerebral ischemia. C57/BL6 mice were subjected to transient middle cerebral artery occlusion (tMCAO) and treated for 5 days with Vehicle or ALK (10 or 25 mg/kg per day via intragastric administration), whereas Sham-operated animals served as controls. Treatment with ALK significantly improved neurological deficits, infarct volume, brain water content and Nissl bodies after stroke (P < 0.05), which did not affect systemic blood pressure. Furthermore, the protection of ALK was also related to decreased levels of apoptosis in mice by enhanced activation of phosphatidylinositol 3-kinase (PI3K)/AKT pathway, increased level of Bcl-2 and reduced Bax expression (P < 0.05). In addition, ALK’s effects were reversed by PI3K inhibitors LY294002 (P < 0.05). Our data indicated that ALK protected the brain from reperfusion injuries without affecting blood pressure, and this effect may be through PI3K/AKT signaling pathway.     

ALK5 and ALK1 Play Antagonistic Roles in Transforming Growth Factor β-Induced Podosome Formation in Aortic Endothelial Cells

Transforming growth factor β (TGF-β) and related cytokines play a central role in the vascular system. In vitro, TGF-β induces aortic endothelial cells to assemble subcellular actin-rich structures specialized for matrix degradation called podosomes. To explore further this TGF-β-specific response and determine in which context podosomes form, ALK5 and ALK1 TGF-β receptor signaling pathways were investigated in bovine aortic endothelial cells. We report that TGF-β drives podosome formation through ALK5 and the downstream effectors Smad2 and Smad3. Concurrent TGF-β-induced ALK1 signaling mitigates ALK5 responses through Smad1. ALK1 signaling induced by BMP9 also antagonizes TGF-β-induced podosome formation, but this occurs through both Smad1 and Smad5. Whereas ALK1 neutralization brings ALK5 signals to full potency for TGF-β-induced podosome formation, ALK1 depletion leads to cell disturbances not compatible with podosome assembly. Thus, ALK1 possesses passive and active modalities. Altogether, our results reveal specific features of ALK1 and ALK5 signaling with potential clinical implications.     

Design,synthesis and biological activity evaluation of novel 4-((1-cyclopropyl-3-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-4-yl) oxy) pyridine-2-yl) amino derivatives as potent transforming growth factor-β (TGF-β) type I receptor inhibitors

TGF-β type I receptor (also known as activin-like kinase 5 or ALK5) plays a critical role in the progression of fibrotic diseases and tumor invasiveness and metastasis, as well. The development of small inhibitors targeting ALK5 has been validated as a potential therapeutic strategy for fibrotic diseases and cancer. Here, we developed various 4-((1-cyclopropyl-3-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-4-yl) oxy) pyridine-2-yl) amino derivatives as ALK5 inhibitors. The optimization led to identification of potent and selective ALK5 inhibitors 12r. The compound 12r exhibited strong inhibitory activity both in vitro and in vivo, and pharmacokinetics study showed an oral bioavailability of 57.6%. Thus, compound 12r may provide as new therapeutic option as ALK5 TGF-βR1 inhibitor.     

Critical roles of the TGF-β type I receptor ALK5 in perichondrial formation and function, cartilage integrity, and osteoblast differentiation during growth plate development

TGF-β has been implicated in the proliferation and differentiation of chondrocytes and osteoblasts. However, the in vivo function of TGF-β in skeletal development is unclear. In this study, we investigated the role of TGF-β signaling in growth plate development by creating mice with a conditional knockout of the TGF-β type I receptor ALK5 (ALK5^CKO) in skeletal progenitor cells using Dermo1-Cre mice. ALK5^CKO mice had short and wide long bones, reduced bone collars, and trabecular bones. In ALK5^CKO growth plates, chondrocytes proliferated and differentiated, but ectopic cartilaginous tissues protruded into the perichondrium. In normal growth plates, ALK5 protein was strongly expressed in perichondrial progenitor cells for osteoblasts, and in a thin chondrocyte layer located adjacent to the perichondrium in the peripheral cartilage. ALK5^CKO growth plates had an abnormally thin perichondrial cell layer and reduced proliferation and differentiation of osteoblasts. These defects in the perichondrium likely caused the short bones and ectopic cartilaginous protrusions. Using tamoxifen-inducible Cre-ER™-mediated ALK5-deficient primary calvarial cell cultures, we found that TGF-β signaling promoted osteoprogenitor proliferation, early differentiation, and commitment to the osteoblastic lineage through the selective MAPKs and Smad2/3 pathways. These results demonstrate the important roles of TGF-β signaling in perichondrium formation and differentiation, as well as in growth plate integrity during skeletal development.     

Fluorescence In Situ Hybridization and Immunohistochemistry as Diagnostic Methods for ALK Positive Non-Small Cell Lung Cancer Patients

Background

Anaplastic Lymphoma Kinase (ALK) positivity represents a novel molecular target in a subset of Non-Small Cell Lung Cancers (NSCLC). We explore Fluorescence in situ Hybridization (FISH) and Immunohistochemistry (IHC) as diagnostic methods for ALK positive patients and to describe its prevalence and outcomes in a population of NSCLC patients.

Methods

NSCLC patients previously screened for Epidermal Growth Factor Receptor (EGFR) at our institution were selected. ALK positive patients were identified by FISH and the value of IHC (D5F3) was explored.

Results

ninety-nine patients were identified. Median age was 61.5 years (range 35–83), all were caucasians, eighty percent were adenocarcinomas, fifty-one percent were male and thirty-eight percent were current smokers. Seven (7.1%) patients were ALK positive by FISH, thirteen (13.1%) were EGFR mutant, and 65 (65.6%) were negative/Wild Type (WT) for both ALK and EGFR. ALK positivity and EGFR mutations were mutually exclusive. ALK positive patients tend to be younger than EGFR mutated or wt patients. ALK positive patients were predominantly never smokers (71.4%) and adenocarcinoma (71.4%). ALK positive and EGFR mutant patients have a better outcome than negative/WT. All patients with ALK FISH negative tumours were negative for ALK IHC. Out of 6 patients positive for ALK FISH with more tissue available, 5 were positive for ALK IHC and 1 negative.

Conclusions

ALK positive patients represent 7.1% of a population of selected NSCLC. ALK positive patients have different clinical features and a better outcome than EGFR WT and ALK negative patients. IHC is a promising method for detecting ALK positive NSCLC patients.     

Hydrogen bonding analysis of phosphoric acid–N,N-dimethylformamide mixtures

Crizotinib is the most effective and the only drug that has been approved for the treatment of anaplastic lymphoma kinase (ALK)-positive lung cancer. Reports suggest that there is a development of an acquired resistance against crizotinib action due to the emergence of several mutations in the ALK gene and F1174L is one such mutation. In this study, we used molecular docking and molecular dynamics (MD) approach to decipher the effect of F1174L mutation in drug–target binding. Docking results suggest that crizotinib was found to adopt the most promising conformations to the native-type ALK by identifying the M1199 residue as a prospective partner for making a hydrogen bond as compared to the mutant-type ALK. MD results showed that the average atom, especially atoms of the native-type ALK-crizotinib complex, movements were less, displayed less fluctuation, fast convergence of energy, and changes in geometry. This shows the stable binding of crizotinib with the native-type ALK in comparison to the mutant-type ALK. We believe that this study could be useful for the logical design of stronger, more selective, and more consistent ALK inhibitor against drug-resistant F1174L mutation.     

Phosphorylation of ΔNp63α via a Novel TGFβ/ALK5 Signaling Mechanism Mediates the Anti-Clonogenic Effects of TGFβ

Genetic analysis of TP63 implicates ΔNp63 isoforms in preservation of replicative capacity and cellular lifespan within adult stem cells. ΔNp63α is also an oncogene and survival factor that mediates therapeutic resistance in squamous carcinomas. These diverse activities are the result of genetic and functional interactions between TP63 and an array of morphogenic and morphostatic signals that govern tissue and tumor stasis, mitotic polarity, and cell fate; however the cellular signals that account for specific functions of TP63 are incompletely understood. To address this we sought to identify signaling pathways that regulate expression, stability or activity of ΔNp63α. An siRNA-based screen of the human kinome identified the Type 1 TGFβ receptor, ALK5, as the kinase required for phosphorylation of ΔNp63α at Serine 66/68 (S66/68). This activity is TGFβ-dependent and sensitive to either ALK5-directed siRNA or the ALK5 kinase inhibitor A83-01. Mechanistic studies support a model in which ALK5 is proteolytically cleaved at the internal juxtamembrane region resulting in the translocation of the C-terminal ALK5-intracellular kinase domain (ALK5^IKD). In this study, we demonstrate that ALK5-mediated phosphorylation of ΔNp63α is required for the anti-clonogenic effects of TGFΒ and ectopic expression of ALK5^IKD mimics these effects. Finally, we present evidence that ultraviolet irradiation-mediated phosphorylation of ΔNp63α is sensitive to ALK5 inhibitors. These findings identify a non-canonical TGFβ-signaling pathway that mediates the anti-clonogenic effects of TGFβ and the effects of cellular stress via ΔNp63α phosphorylation.     

BioID-Screening Identifies PEAK1 and SHP2 as Components of the ALK Proximitome in Neuroblastoma Cells

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase (RTK) that is mutated in approximately 10% of pediatric neuroblastoma (NB). To shed light on ALK-driven signaling processes, we employed BioID-based in vivo proximity labeling to identify molecules that interact intracellularly with ALK. NB-derived SK-N-AS and SK-N-BE(2) cells expressing inducible ALK-BirA* fusion proteins were generated and stimulated with ALKAL ligands in the presence and absence of the ALK tyrosine kinase inhibitor (TKI) lorlatinib. LC/MS-MS analysis identified multiple proteins, including PEAK1 and SHP2, which were validated as ALK interactors in NB cells. Further analysis of the ALK-SHP2 interaction confirmed that the ALK-SHP2 interaction as well as SHP2-Y542 phosphorylation was dependent on ALK activation. Use of the SHP2 inhibitors, SHP099 and RMC-4550, resulted in inhibition of cell growth in ALK-driven NB cells. In addition, we noted a strong synergistic effect of combined ALK and SHP2 inhibition that was specific to ALK-driven NB cells, suggesting a potential therapeutic option for ALK-driven NB.     

Synthesis and biological evaluation of benzo[4,5]imidazo[1,2-c]pyrimidine and benzo[4,5]imidazo[1,2-a]pyrazine derivatives as anaplastic lymphoma kinase inhibitors

Chromosomal translocations involving anaplastic lymphoma kinase (ALK) are the driving mutations for a range of cancers and ALK is thus considered an attractive therapeutic target. We synthesized a series of functionalized benzo[4,5]imidazo[1,2-c]pyrimidines and benzo[4,5]imidazo[1,2-a]pyrazines by an aza-Graebe–Ullman reaction, followed by palladium-catalyzed cross-coupling reactions. A sequential regioselective cross-coupling route is reported for the synthesis of unsymmetrically disubstituted benzo[4,5]imidazo[1,2-a]pyrazines. The inhibition of ALK was evaluated and compound 19 in particular showed good activity against both the wild type and crizotinib-resistant L1196M mutant in vitro and in ALK-transfected BaF3 cells.