Unpacking a Multi-Omics Treasure-Hunt: How Cladribine Re-wires Immune Cells in Multiple Sclerosis

Cladribine is one of only two licensed immune-reconstitution therapies for relapsing multiple sclerosis, yet its molecular mode-of-action and the biomarkers that might predict response remain largely undefined. Fissolo et al. set out to close that gap by performing an integrated transcriptomic, proteomic and micro-RNA (“multi-omics”) analysis of patients’ blood cells exposed to cladribine, with the aim of mapping the drug’s signature and surfacing predictive markers.

How the investigators approached the problem
Peripheral-blood mononuclear cells (PBMCs) from ten untreated MS donors were activated, then incubated for four hours with either no drug, active cladribine, or cladribine whose activation had been blocked by excess deoxycytidine. Whole-genome microarrays, mass-spectrometry proteomics and miRNA arrays captured changes across the three molecular layers. Two complementary bio-informatics pipelines—DIABLO for holistic integration and a step-wise “filter-then-merge” routine for pinpointing individual molecules—were applied. Key candidates were subsequently measured by qPCR in fresh blood from five cladribine-treated patients at baseline, three and twelve months.

What they found
Across all layers cladribine drove a broad suppression of gene, protein and miRNA expression, while the drug rendered inactive by deoxycytidine generated an intermediate profile, suggesting kinase-independent effects. Out of more than 200 dysregulated genes and proteins, exactly four overlapped at both RNA and protein level—PPIF (Cyclophilin-D) and JUN, both down-regulated; NHLRC2 up-regulated; and NIBAN2 down-regulated.

Fifty-five miRNAs changed significantly, but only twenty-two were predicted to target the four flagship genes. Of those, miR-30b-5p, miR-30e-5p and the well-known immune regulator miR-21-5p showed consistent modulation in patient samples: each dipped at three months, with miR-30b-5p remaining low and the other two rebounding by month twelve.

How the pieces fit together
Lower PPIF would reduce Cyclophilin-D–mediated opening of the mitochondrial permeability transition pore, a mechanism whose inhibition preserves axons and speeds recovery in experimental MS models; cladribine’s neuro-protective edge may therefore extend beyond simple lymphocyte depletion.

Conversely, higher NHLRC2—released from the restraint of its inhibitory miR-30b-5p—could enhance macrophage phagocytosis and aid myelin debris clearance, a pre-requisite for remyelination. The staggered miRNA pattern suggests an early anti-inflammatory wave followed by long-term immune remodelling, offering a molecular “clock” by which clinicians might time additional interventions.

Implications for practice and research
If larger cohorts confirm the PPIF ↓ / NHLRC2 ↑ / miR-30b-5p ↓ triad, clinicians could gain a blood-based yardstick of cladribine activity, helping to separate true responders from patients who should switch therapy sooner. More broadly, the work underscores that cladribine is not merely cytotoxic; even its inactive form nudges distinct signalling pathways, hinting at rational combination strategies (for example pairing with targeted mitochondrial-pore inhibitors to amplify neuroprotection).

Caveats to keep in mind
The analysis rests on a small sample (ten donors in vitro, five patients ex vivo), the four-hour culture window may miss later transcriptional waves, and PBMCs are a surrogate for the central nervous system milieu where MS pathology unfolds.

Where next?
Multi-centre longitudinal studies should validate these candidate biomarkers, ideally with single-cell multi-omics to resolve cell-type specificity and cerebrospinal-fluid profiling to link peripheral signatures with central lesions.

Take-home message
Cladribine leaves a concise, multi-layered molecular footprint—defined by PPIF, NHLRC2, JUN, NIBAN2 and a set of regulatory miRNAs—that can be tracked in patient blood. Decoding that footprint not only clarifies how the drug works but also lights the path toward personalised monitoring of immune-reconstitution therapies in multiple sclerosis.

Disclaimer: This blog post is based on the provided research article and is intended for informational purposes only. It is not intended to provide medical advice. Please consult with a healthcare professional for any health concerns.

References:
Fissolo, N., Calvo-Barreiro, L., Eixarch, H., Boschert, U., Villar, L. M., Costa-Frossard, L., ... & Comabella, M. (2023). Molecular signature associated with cladribine treatment in patients with multiple sclerosis. Frontiers in immunology, 14, 1233546.