EML4-ALK Variant 3 Promotes Mitotic Errors and Spindle Assembly Checkpoint Deficiency Leading to Increased Microtubule Poison Sensitivity

Abstract

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.

Loss of Spindly sensitizes EML4-ALK v3 lung cancer cells to HSP90 inhibitors

“…In contrast to v1, EML4-ALK v3 localizes to micro-tubules (5; 6) and might be associated, directly or indirectly, with other proteins involved in the formation of the mitotic spindle. For this reason and because of the high-confidence score of the SPDL1 gene in v3 H2228 cells, we further investigated the role played by depletion of Spindly in increasing the sensitivity of EML4-ALK v3 NSCLC cells to low concentrations of HSP90i.…”

“…We observed loss of cellular fitness upon combined depletion of Spindly and prolonged exposure to low concentrations of HSP90i exclusively in EML4-ALK v3, and not in v1, NSCLC cells. It has been shown that while both v1 and v3 localize to cytoplasmic granules, only v3 binds to microtubules (5; 6; 7). It is tempting to speculate that v3 may bring the tyrosine kinase activity of ALK to the spindle where the phosphorylation of proteins involved in chromosome congression might generate a specific vulnerability to loss of Spindly and to prolonged treatment with low concentrations of HSP90i.…”

“…EML4-ALK v1 and EML4-ALK v3 are the most common variants and account for the majority of all cases (4). The longer v1 localizes to cytoplasmic granules and retains a portion of the tandem atypical propeller in EMLs (TAPE) domain of EML4, which confers structural instability; in contrast, the shorter v3 does not retain portions of the TAPE domain, is more stable and localizes to both cytoplasmic granules and microtubules (5; 6; 7). All variants retain the trimerization domain of EML4, which leads to the cancer-addicting constitutive activation of ALK and downstream MAPK/PI3K signaling.…”

Alternative Treatment Options to ALK Inhibitor Monotherapy for EML4-ALK-Driven Lung Cancer

“…Furthermore, they found that the EML4-ALK V3 protein localised to the mitotic spindle microtubules with cells exhibiting stabilized K-fibres and increased mitotic defects including multipolar spindles, lagging chromosomes in metaphase and misaligned chromosomes in anaphase compared to cells expressing V1. Finally, the activity of the spindle assembly checkpoint was found to be reduced in cells expressing V3 compared to V1 [ 109 ]. Taken together, these two studies demonstrate the potential of combining ALK TKIs with microtubule poisons or targeted mitotic agents as an alternative to ALK inhibitor monotherapy ( Figure 5 ).…”

“…Microtubule poisons have been regularly used as chemotherapies for many tumour types, including One example is vincristine, a vinca alkaloid chemotherapy that promotes microtubule depolymerization by binding to tubulin monomers [107]. Sampson et al (2022) sought to test the benefit of combining vincristine with ALK inhibitors in EML4-ALK patient-derived cells. They found that the combination of vincristine together with crizotinib or ceritinib led to an increased loss of cell viability and enhanced apoptosis in cells expressing V1, but not V3 [108].…”