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

The second most common malignancy in both men and women is lung cancer. Smoking is the primary cause of lung cancer risk factors, accounting for 75-80% of lung cancer-related fatalities. Small-cell carcinomas and non-small-cell carcinomas are the two types of lung cancer that may be generically categorized. Up to 75% of lung cancer cases are non-small-cell lung cancer, which is more prevalent. The oncogenic fusion protein EML4-ALK, which results from a chromosomal inversion event on the short arm of chromosome 2, is responsible for 5% of lung adenocarcinomas, according to a study on molecular profiling of these tumours. Current best practices for treating patients with ALK-positive NSCLC include the use of targeted ALK inhibitors such as crizotinib, ceritinib, alectinib, and lorlatinib. In contrast, the TAPE domain interacts to soluble /-tubulin heterodimers, the N-terminal TD and basic linker of the microtubule-associated protein EML4 links to microtubules. Although the exact mechanism for how EML4 promotes microtubule stability is yet unknown. An oncogenic fusion protein called EML4-ALK is found in around 5% of non-small cell lung tumors (NSCLC). Alternative breakpoints produce diverse variations in the gene encoding EML4, and these variants are associated with radically different patient outcomes.

Compared to patients with other prevalent variants, such V1, people with EML4-ALK variant 3 (V3) respond poorly to ALK inhibitors and have worse survival rates. In a new study published in Molecular Cancer Research, scientist Kellie Lucken, Laura O’Regan, Sarah Pashley, Sam Khan and Professor Andrew Fry from the University of Leicester together with Professor Jene Choi at University of Ulsan College of Medicine and Dr.  Josephina Sampson and Professor Richard Bayliss at University of Leeds demonstrated that EML4-ALK V3 but not V1 causes hyperstabilized K-fibers in mitosis in addition to errors in chromosome congression and segregation using 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).

The research team showed that expression of EML4-ALK V3 but not V1 leads to a variety of mitotic abnormalities, including the production of multipolar spindles, misaligned chromosomes, and lagging chromosomes in patients with ALK-positive NSCLC cells and isogenic recombinant Beas-2B cells. In interphase cells expressing V3, micronuclei and amplified centrosomes were also seen more frequently. These findings might be related to incomplete cell division as well as unbalanced centrosome and chromosomal segregation. Researchers found that the bulk of these mitotic mistakes are independent of the enzymatic activity of ALK using a particular pharmacological inhibitor of ALK, TAE-684. They discovered that cells expressing EML4- ALK V3 failed to stop in mitosis and recruited less BubR1 to kinetochores when treated with the microtubule poisons nocodazole and paclitaxel, in addition to abnormalities in spindle structure and chromosomal congression and segregation. The authors suggested that SAC activity is diminished in EML4-ALK V3 cells. In cells expressing EML4-ALK V3, abnormal microtubule dynamics and trafficking lead to kinetochore assembly disruption. Because cells expressing a kinase-inactive version of EML4-ALK V3 showed a stronger response from the SAC, researchers discovered that ALK activity may be partially to blame for the disruption of the SAC.

Additionally, they verified in H2228 cells that TAE-684 therapy or EML4-ALK V3 deletion extended mitosis in comparison to untreated cells, confirming the idea that ALK activity inhibits SAC function. The authors found that treating EML4-ALK V3-expressing H2228 cells with the microtubule-stabilizing drug paclitaxel in conjunction with therapeutically authorised ALK inhibitors resulted in a synergistic loss of cell viability. H2228 cells responded synergistically with paclitaxel across a range of dosages of alectinib, ceretinib, and crizotinib, demonstrating that combination therapies promote apoptotic cell death in both H3122 and H2228 cells.

In summary, patients with EML4-ALK V3 driven cancers have an urgent medical need, and the research presented here adds to the body of evidence supporting the possibility that a polytherapy approach combining ALK inhibitors with, in this case, paclitaxel, could be successful and lessen the risk of developing acquired resistance. According to authors, paclitaxel, a microtubule poison, combined with specific ALK inhibitors may provide NSCLC patients with tumours expressing the EML4-ALK V3 oncogenic fusion an efficient new therapy option.