Search

Anaplastic lymphoma kinase (ALK) can be driven to oncogenic activity by different types of mutational events such as point‐mutations, for example F1174L in neuroblastoma, and gene fusions, for example with echinoderm microtubule‐associated protein‐like 4 (EML4) in non‐small cell lung cancer (NSCLC). EML4‐ALK variants result from different breakpoints, generating fusions of different sizes and properties. The most common variants (Variant 1 and Variant 3) form cellular compartments with distinct physical properties. The presence of a partial, probably misfolded beta‐propeller domain in variant 1 confers solid‐like properties to the compartments it forms, greater dependence on Hsp90 for protein stability and higher cell sensitivity to ALK tyrosine kinase inhibitors (TKIs). These differences translate to the clinic because variant 3, on average, worsens patient prognosis and increases metastatic risk. Latest generation ALK‐TKIs are beneficial for most patients with EML4‐ALK fusions. However, resistance to ALK inhibitors can occur via point‐mutations within the kinase domain of the EML4‐ALK fusion, for example G1202R, reducing inhibitor effectiveness. Here, we discuss the biology of EML4‐ALK variants, their impact on treatment response, ALK‐TKI drug resistance mechanisms and potential combination therapies. [ABSTRACT FROM AUTHOR]

Immune checkpoint inhibitors (ICIs) are utilised in treating non‐small cell lung cancer (NSCLC) by enhancing the immune response against cancer cells. However, they are not effective against cancers with certain genetic alterations. A recent study by Mota et al. focussed on understanding why ALK+ NSCLC cancers are immune cold and making them more receptive to ICIs using a vaccine‐based approach. The study highlighted cell‐specific differences in the presentation of immunogenic peptides and the location of tumours as factors in the poor immune response. Vaccines based on ALK peptides improved immune response, and when combined with ICIs, this led to a striking improvement in survival in a mouse model of ALK+ NSCLC. [ABSTRACT FROM AUTHOR]

Hsp72 is a member of the 70-kDa heat shock family of molecular chaperones (Hsp70s) that comprise a nucleotide-binding domain (NBD) and a substrate-binding domain (SBD) connected by a linker that couples the exchange of adenosine diphosphate (ADP) for adenosine triphosphate (ATP) with the release of the protein substrate. Mitotic phosphorylation of Hsp72 by the kinase NEK6 at Thr66 located in the NBD promotes the localization of Hsp72 to the mitotic spindle and is required for efficient spindle assembly and chromosome congression and segregation. We determined the crystal structure of the Hsp72 NBD containing a genetically encoded phosphoserine at position 66. This revealed structural changes that stabilized interactions between subdomains within the NBD. ATP binding to the NBD of unmodified Hsp72 resulted in the release of substrate from the SBD, but phosphorylated Hsp72 retained substrate in the presence of ATP. Mutations that prevented phosphorylation-dependent subdomain interactions restored the connection between ATP binding and substrate release. Thus, phosphorylation of Thr66 is a reversible mechanism that decouples the allosteric connection between nucleotide binding and substrate release, providing further insight into the regulation of the Hsp70 family. We propose that phosphorylation of Hsp72 on Thr66 by NEK6 during mitosis promotes its localization to the spindle by stabilizing its interactions with components of the mitotic spindle. [ABSTRACT FROM AUTHOR]

ABSTRACT We describe a consanguineous Iraqi family with Leber congenital amaurosis ( LCA), Joubert syndrome ( JBTS), and polycystic kidney disease ( PKD). Targeted next-generation sequencing for excluding mutations in known LCA and JBTS genes, homozygosity mapping, and whole-exome sequencing identified a homozygous missense variant, c.317 G> C (p. Arg106 Pro), in POC1 B, a gene essential for ciliogenesis, basal body, and centrosome integrity. In silico modeling suggested a requirement of p. Arg106 for the formation of the third WD40 repeat and a protein interaction interface. In human and mouse retina, POC1 B localized to the basal body and centriole adjacent to the connecting cilium of photoreceptors and in synapses of the outer plexiform layer. Knockdown of Poc1b in zebrafish caused cystic kidneys and retinal degeneration with shortened and reduced photoreceptor connecting cilia, compatible with the human syndromic ciliopathy. A recent study describes homozygosity for p. Arg106 Pro POC1B in a family with nonsyndromic cone-rod dystrophy. The phenotype associated with homozygous p. Arg106 Pro POC1B may thus be highly variable, analogous to homozygous p. Leu710 Ser in WDR19 causing either isolated retinitis pigmentosa or Jeune syndrome. Our study indicates that POC1 B is required for retinal integrity, and we propose POC1 B mutations as a probable cause for JBTS with severe PKD. [ABSTRACT FROM AUTHOR]

Background: Anaplastic lymphoma kinase (ALK)-targeted tyrosine kinase inhibitors (TKIs) improve patient survival; however, some patients develop ALK-TKI resistance with unidentified mechanisms. We investigated ErbB family and c-MET expression in patients with ALK-positive non-small cell lung cancer (NSCLC) to understand their roles in the ALK-TKI response., Methods: We studied 72 patients with advanced ALK-positive NSCLC with EML4-ALK fusion variant subtyping and immunostaining for c-MET, EGFR, HER2, and HER3 on tissue specimens both pre- (primary) and post-treatment (secondary) with ALK-TKI. We investigated the association of their expression with survival outcomes and assessed the effectiveness of combining ALK and EGFR inhibitors in ALK-positive NSCLC cell lines stimulated with the HER3-specific ligand HRG1., Results: High expression of c-MET, EGFR, HER2, and HER3 was observed in 4.9%, 18.0%, 1.6%, and 25.8% of primary tumors, respectively, and 18.5%, 37.0%, 10.7%, and 35.7% of secondary tumors, respectively. HER3 overexpression in primary tumors showed inferior survival (P=0.132). In the subgroup with EML4-ALK variant 1/2 (V1/V2), HER3 overexpression was significantly associated with inferior survival in both primary and secondary tumors (P=0.022 and P=0.004, respectively). Combination treatment with lorlatinib and erlotinib significantly reduced HRG1-induced activation of RTK signaling in ALK-positive NSCLC cells., Conclusions: HER3 overexpression has potential as a prognostic marker in ALK-positive NSCLCs, including ALK-TKI naïve and treated cases, especially those with EML4-ALK V1/V2. Assessing HER3 expression may be crucial for treatment planning and outcome prediction in these patients., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-23-804/coif). The authors have no conflicts of interest to declare., (2024 Translational Lung Cancer Research. All rights reserved.)

EML4-ALK is an oncogenic fusion protein present in approximately 5% of non-small cell lung cancers (NSCLC). Alternative breakpoints in the gene encoding EML4 result in distinct variants that are linked to markedly different patient outcomes. Patients with EML4-ALK variant 3 (V3) respond poorly to ALK inhibitors and have lower survival rates compared with patients with other common variants, such as V1. Here, we use isogenic Beas-2B bronchial epithelial cell lines expressing EML4-ALK V1 or V3, as well as ALK-positive NSCLC patient cells that express V1 (H3122 cells) or V3 (H2228 cells), to show that EML4-ALK V3 but not V1 leads to hyperstabilized K-fibers in mitosis, as well as errors in chromosome congression and segregation. This is consistent with our observation that EML4-ALK V3 but not V1 localizes to spindle microtubules and that wild-type EML4 is a microtubule stabilizing protein. In addition, cells expressing EML4-ALK V3 exhibit loss of spindle assembly checkpoint control that is at least in part dependent on ALK catalytic activity. Finally, we demonstrate that cells expressing EML4-ALK V3 have increased sensitivity to microtubule poisons that interfere with mitotic spindle assembly, whereas combination treatment with paclitaxel and clinically approved ALK inhibitors leads to a synergistic response in terms of reduced survival of H2228 cells., Implications: This study suggests that combining the microtubule poison, paclitaxel, with targeted ALK inhibitors may provide an effective new treatment option for patients with NSCLC with tumors that express the EML4-ALK V3 oncogenic fusion., (©2022 The Authors; Published by the American Association for Cancer Research.)

The oncogenic fusion of EML4-ALK is present in about 4-6% of non-small cell lung cancer (NSCLC). A targeted approach with ALK tyrosine kinase inhibitors (TKIs) has been proven highly effective in ALK-positive NSCLC patients. However, despite the initial responses, the outcome of the treatment is variable. Previous studies have shown that the differential response depends in part on the type of EML4-ALK variant. Here, we examined the combination of ALK inhibitors and microtubule poison, vincristine, in cells expressing EML4-ALK V1 and V3, the two most common variants in NSCLC. We showed that combination therapy of ALK-TKIs with vincristine had anti-proliferative effects and blocked RAS/MAPK, PI3K/AKT and JAK/STAT3 signalling pathways in EML4-ALK V1 but not V3 cells. Our results demonstrate that high levels of tubulin acetylation are associated with poor response to vincristine in EML4-ALK V3 cells. Additionally, we demonstrated differences in microtubule stability between the two EML4-ALK fusions. EML4-ALK V3 cells exhibited dynamic microtubules that confer poor response to vincristine compared to V1 cells. Hence, we suggested that the portion of EML4 in the fusion has an important role for the outcome of the combination treatment.

Cancer cells frequently possess extra amplified centrosomes clustered into two poles whose pseudo-bipolar spindles exhibit reduced fidelity of chromosome segregation and promote genetic instability. Inhibition of centrosome clustering triggers multipolar spindle formation and mitotic catastrophe, offering an attractive therapeutic approach to selectively kill cells with amplified centrosomes. However, mechanisms of centrosome clustering remain poorly understood. Here, we identify a new pathway that acts through NIMA-related kinase 6 (Nek6) and Hsp72 to promote centrosome clustering. Nek6, as well as its upstream activators polo-like kinase 1 and Aurora-A, targeted Hsp72 to the poles of cells with amplified centrosomes. Unlike some centrosome declustering agents, blocking Hsp72 or Nek6 function did not induce formation of acentrosomal poles, meaning that multipolar spindles were observable only in cells with amplified centrosomes. Inhibition of Hsp72 in acute lymphoblastic leukemia cells resulted in increased multipolar spindle frequency that correlated with centrosome amplification, while loss of Hsp72 or Nek6 function in noncancer-derived cells disturbs neither spindle formation nor mitotic progression. Hence, the Nek6-Hsp72 module represents a novel actionable pathway for selective targeting of cancer cells with amplified centrosomes. Cancer Res; 77(18); 4785-96. ©2017 AACR ., (©2017 American Association for Cancer Research.)

The centrosome is an unusual organelle that lacks a surrounding membrane, raising the question of what limits its size and shape. Moreover, while electron microscopy (EM) has provided a detailed view of centriole architecture, there has been limited understanding of how the second major component of centrosomes, the pericentriolar material (PCM), is organized. Here, we summarize exciting recent findings from super-resolution fluorescence imaging, structural biology, and biochemical reconstitution that together reveal the presence of ordered layers and complex gel-like scaffolds in the PCM. Moreover, we discuss how this is leading to a better understanding of the process of microtubule nucleation, how alterations in PCM size are regulated in cycling and differentiated cells, and why mutations in PCM components lead to specific human pathologies., Competing Interests: Competing interests: The authors declare that they have no competing interests.No competing interests were disclosed.No competing interests were disclosed.No competing interests were disclosed.

Hsp70 proteins represent a family of chaperones that regulate cellular homeostasis and are required for cancer cell survival. However, their function and regulation in mitosis remain unknown. In this paper, we show that the major inducible cytoplasmic Hsp70 isoform, Hsp72, is required for assembly of a robust bipolar spindle capable of efficient chromosome congression. Mechanistically, Hsp72 associates with the K-fiber-stabilizing proteins, ch-TOG and TACC3, and promotes their interaction with each other and recruitment to spindle microtubules (MTs). Targeting of Hsp72 to the mitotic spindle is dependent on phosphorylation at Thr-66 within its nucleotide-binding domain by the Nek6 kinase. Phosphorylated Hsp72 concentrates on spindle poles and sites of MT-kinetochore attachment. A phosphomimetic Hsp72 mutant rescued defects in K-fiber assembly, ch-TOG/TACC3 recruitment and mitotic progression that also resulted from Nek6 depletion. We therefore propose that Nek6 facilitates association of Hsp72 with the mitotic spindle, where it promotes stable K-fiber assembly through recruitment of the ch-TOG-TACC3 complex., (© 2015 O’Regan et al.)