Upregulation of FOXM1 leads to diminished drug sensitivity in myeloma

Abstract

Background

Building upon prior foundational research that unequivocally demonstrated the significant involvement of the transcription factor forkhead box M1 (FOXM1) in shaping the biological characteristics and influencing the clinical outcome of a particularly high-risk subset of patients diagnosed with newly diagnosed multiple myeloma (nMM), the present study was meticulously designed to expand this understanding. A central premise for this investigation was to evaluate whether the gene expression of FOXM1 undergoes further, potentially detrimental, upregulation specifically upon tumor recurrence in patients who have progressed to relapsed multiple myeloma (rMM). This exploration into the dynamic changes of FOXM1 expression in the context of disease progression is crucial, as the acquisition of drug resistance is a hallmark of relapsed disease, posing significant challenges to effective treatment. Concurrently, a second pivotal objective of this study was to rigorously assess the compelling hypothesis that elevated levels of FOXM1, whether inherently present or acquired during disease progression, actively contribute to a diminished sensitivity of myeloma cells to commonly employed and otherwise effective anti-myeloma therapeutic agents. Particular focus was placed on two cornerstone drugs in multiple myeloma therapy: bortezomib (Bz), a highly effective proteasome inhibitor, and doxorubicin (Dox), a potent DNA intercalator. Understanding the mechanisms of resistance to these critical agents is vital for improving patient outcomes.

Methods

To systematically address the stated research objectives, a robust and comprehensive methodological framework was implemented. The quantitative assessment of FOXM1 messenger RNA (mRNA) levels was performed on an extensive cohort of 88 meticulously paired myeloma samples, with each pair comprising specimens obtained from individual patients at both their initial diagnosis of nMM and subsequently upon tumor recurrence, representing their rMM state. Gene expression microarrays served as the primary and highly sensitive measurement tool for this extensive molecular profiling, enabling a broad and unbiased survey of gene activity. Rigorous statistical methods were then applied to these vast datasets to precisely identify sources of differential gene expression between the paired samples and to conduct in-depth outlier analyses, thereby highlighting individual cases with exceptionally marked changes in FOXM1 expression. Complementing the patient sample analysis, an innovative *in vitro* cellular model was developed utilizing two independent human myeloma cell lines (HMCLs). These cell lines were specifically engineered to either maintain normal, endogenous levels of FOXM1 (designated FOXM1N) or to stably express significantly elevated levels of FOXM1 through the introduction of lentivirus-encoded FOXM1 (designated FOXM1Hi). This controlled cellular system provided an invaluable platform to directly ascertain FOXM1-dependent alterations in key cellular behaviors, including cell proliferation rates, overall cellular survival, the activity of drug efflux pumps (which are often implicated in drug resistance), and, critically, their inherent sensitivity to various anti-myeloma drugs. Furthermore, to probe the molecular cascades downstream of FOXM1, the expression levels of the retinoblastoma (Rb) protein, a pivotal tumor suppressor and cell cycle regulator, were precisely determined with the assistance of Western blotting, a gold-standard technique for protein quantification.

Results

The meticulous analysis of gene expression in the paired patient samples yielded highly significant findings, revealing that a substantial proportion of patients exhibited an upregulation of FOXM1 upon tumor recurrence. Specifically, FOXM1 upregulation was observed in 61 out of the 88 (representing 69%) patients with relapsed multiple myeloma. Even more striking, a subset of 4 patients within this cohort demonstrated exceptionally pronounced increases, exhibiting greater than 20-fold elevated expression peaks of FOXM1 mRNA, underscoring the potential severity of this transcriptional dysregulation in advanced disease. Further investigation using the engineered human myeloma cell lines provided direct mechanistic evidence for the functional consequences of elevated FOXM1. Increased FOXM1 levels in the FOXM1Hi myeloma cells demonstrably caused a partial but significant resistance *in vitro* to both bortezomib and doxorubicin, when compared to the control FOXM1N myeloma cells. Specifically, resistance factors ranged from 1.9- to 5.6-fold for bortezomib and 1.5- to 2.9-fold for doxorubicin, indicating that the presence of high FOXM1 levels compromises the efficacy of these cornerstone agents. The clinical relevance of these *in vitro* findings was powerfully corroborated by *in vivo* studies using myeloma-in-mouse xenograft models, which confirmed the reduced sensitivity of FOXM1Hi cells to bortezomib, thereby solidifying the translational implications of FOXM1-mediated resistance. Furthermore, the mechanistic studies unveiled a critical link between FOXM1 and the retinoblastoma protein. FOXM1-dependent regulation of both total and phosphorylated forms of Rb was observed, providing compelling evidence that aligns with a sophisticated working model of myeloma pathogenesis. This model suggests that FOXM1 plays a dual role in governing both chromosomal instability (CIN), a major driver of genomic aberrations in cancer, and E2F-dependent proliferation, which promotes uncontrolled cell division. These critical cellular processes are mechanistically orchestrated through specific interactions: FOXM1′s role in CIN involves its interaction with NIMA related kinase 2 (NEK2), while its influence on E2F-dependent proliferation is mediated through an interaction with cyclin dependent kinase 6 (CDK6).

Conclusions

In conclusion, the comprehensive findings from this study have significantly enhanced our understanding of the intricately emerging FOXM1 genetic network within the complex landscape of multiple myeloma biology and progression. The clear demonstration of FOXM1 upregulation upon relapse and its direct causal link to drug resistance provides critical insights into the mechanisms underlying therapeutic failure in a significant proportion of patients. Crucially, this research provides robust preclinical support for the innovative therapeutic strategy of directly targeting the FOXM1-NEK2 pathway and the CDK4/6-Rb-E2F axis. This can be achieved through the judicious application of specific small-molecule inhibitors designed to antagonize the activity of CDK and NEK2 kinases. BMS-265246 These findings lay a solid foundation for the rational development of novel therapeutic interventions. Therefore, rigorous and well-designed clinical research is unequivocally warranted to assess whether this promising pharmacological approach, by selectively inhibiting these FOXM1-regulated pathways, can effectively overcome drug resistance in patients with FOXM1Hi myeloma. Ultimately, the successful translation of these preclinical insights into clinical practice holds the immense potential to significantly improve the long-term outcome and prognosis for patients battling multiple myeloma, particularly those in whom this critical transcription factor is expressed at detrimental high levels, representing a challenging and high-risk patient population.

Keywords: Cellular senescence; Plasma-cell neoplasm; Small-drug inhibitor; Targeted cancer therapy.