Treatment failure in malignant diseases, including multiple myeloma (MM), is due to drug resistance of a minor population of cancer cells, already present de novo. Gene expression profiling (GEP) data can be obtained repeatedly during different phases of treatment. Thus, it is possible to monitor newly acquired genetic changes in the drug-resistant MM clone.
Nineteen primary MM patients enrolled in protocol 25009, provided 59 samples, obtained at baseline, pre-1st, pre-2nd, post-2nd stem cell transplant, and in consolidation. GEP was performed using Affymetrix U133Plus2 chips. In addition, a total of 2552 samples with GEP and clinical data, obtained from published studies, were analyzed. We induced NEK2 over-expression and knockdown using a lentiviral delivery system. A NEK2 inhibitor, NI-2, was identified by screening a kinase inhibitor library including 160 compounds and was used for our in vitro and in vivo studies.
By comparing serial GEP samples at baseline versus after treatment or at rapid relapse, we defined 56 genes associated with drug resistance. Ten of the top 20 genes belong to the well established chromosomal instability (CIN) signature. The mean expression levels of CIN genes define a high-risk score present in 10% of newly diagnosed MM patients, which is associated with shorter durations of response, event-free (EFS) (HR = 4.8; P < 0.001), and overall survival (OS)(HR = 5.2; P < 0.001). NEK2 is a serine-threonine kinase and a CIN gene. It was the most significant of the 56 genes related to drug resistance. We found that high expression of NEK2 was associated with a shorter EFS and OS in multiple other cancers, such as breast, lung, AML, mesothelioma, glioma and mantle cell lymphoma. As expected, NEK2 over-expression induced CIN in MM cells evidenced by array-CGH. Over-expressing NEK2 in multiple cancer cell lines resulted in enhanced cell proliferation and increased drug resistance, whereas knockdown of NEK2 by sh RNA induced cancer cell death and growth inhibition. NI-2, a NEK2 kinase inhibitor, significantly suppressed NEK2 activity and induced cancer cell apoptosis, most pronounced in cancer cells with induced NEK2 over-expression. Our in vivo studies in the 5TGM1 C57BL/KaLwRij myeloma mouse model confirm that NI-2 treatment indeed overcomes drug resistance and extends mouse survival also drug-resistant 5GM1 cells were infused (P < 0.01). NI-2 showed a synergistic killing effect on MM cells when combined with bortezomib.
We conclude that NEK2 is an important indicator of drug resistance, and therefore, of poor prognosis in MM and many other cancers. It represents a logical target to overcome drug resistance. In combination with other chemotherapies, it may result in the curing of previously incurable neoplasias, including MM. Our future work will aim at modifying NEK2 inhibitors to optimize activity and decrease toxicity of NEK2 kinase inhibitors in collaboration with our medicinal chemistry colleagues.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.