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Multiple Myeloma Therapy Targets, Resistance Pathways Identified With Single-Cell Sequencing

NEW YORK – After conducting a prospective, multicenter, single-arm clinical trial of multiple myeloma (MM) combined with a longitudinal single-cell RNA-sequencing (scRNA-seq) study of the disease's molecular underpinnings, researchers in Israel identified new molecular pathways of resistance, including hypoxia tolerance, protein folding, and mitochondria respiration.

In a paper published on Monday in Nature Medicine, the researchers noted that disease heterogeneity within and between treatment-resistant MM patients is poorly characterized. They found new pathways of treatment resistance in prespecified exploratory analyses of their clinical trial while conducting a comparison of 41 primary refractory and early relapsed MM patients with 11 healthy subjects and 15 newly diagnosed MM patients.

The researchers also found that peptidylprolyl isomerase A (PPIA), a central enzyme in the protein-folding response pathway, could serve as a potential new target for treatment-resistant MM. Deletion of PPIA using CRISPR-Cas9 or inhibition of PPIA using the small molecule inhibitor ciclosporin significantly sensitized MM tumor cells to proteasome inhibitors.

"Together, our study defines a roadmap for integrating scRNA-seq in clinical trials, identifies a signature of highly resistant MM patients, and discovers PPIA as a potent therapeutic target for these tumors," the authors wrote.

For the clinical trial, the researchers enrolled 41 newly diagnosed MM patients who had either failed to respond or experienced early relapse after a bortezomib-containing induction regimen. They planned to evaluate the safety and efficacy of a combination therapy containing daratumumab (Janssen's Darzalex), carfilzomib (Onyx Pharmaceuticals' Kyprolis), lenalidomide (Celgene's Revlimid), and dexamethasone. The primary clinical endpoint was safety and tolerability, and secondary endpoints included overall response rate, progression-free survival, and overall survival. Overall, they said treatment was safe and well tolerated. Patients achieved an overall response rate of 88 percent, 13 percent of the cohort achieved a complete response or better, 50 percent achieved a very good partial response, and 25 percent achieved a partial response. Median progression-free survival was 14.7 months for the entire cohort.

In analyzing their single-cell data on these patients, the researchers found that although every patient displayed a unique transcriptional state, they had commonly overexpressed driver genes, such as CCND1, CCND2, and FRZB27. The investigators also detected a small fraction of seemingly healthy polyclonal plasma cells in most patients, with higher variability in the primary refractory MM cohort after the first line of treatment.

Overall, their data defined the molecular signature and driver genes of malignant plasma cells within newly diagnosed MM and primary refractory MM patients, showing no significant difference between the two groups.

They did find that refractory and resistant MM patients were characterized by unique stress pathways, and hypothesized that dedicated molecular pathways defined the resistance mechanisms of malignant plasma cells of the refractory patients. Indeed, an analysis found that 66 diferentially expressed genes distinguished the newly diagnosed from the refractory patients' malignant plasma cells. The genes included some previously described high-risk MM factors such as STMN1, PSMB4, RRM2, and TYMS28-32, as well as new genes not previously associated with high-risk MM, such as PPIA.

"Together, our results describe a novel MM resistance signature that defines a subset of [primary refractory] MM patients, including perturbation in mitochondrial stress genes, the ER and UPR pathway, and proteasome machinery," the authors wrote.

Importantly, when they combined the data from the clinical trial with the analyses from the single-cell study, the researchers found that signatures of the primary refractory MM patients could predict clinical response to the combination treatment.

For example, they detected several candidate markers that were highly differential between the responder and the non-responder patients, including the downregulation of multiple genes associated with plasma cell function. Further, CD38, which is the target of daratumumab, was downregulated in the nonresponsive patient cohort, and non-responders had significantly upregulated levels of the proteasome genes PSMD4 and PSMB4, highlighting potential escape mechanisms from the proteasome inhibitor in the combination regimen.

"Our results demonstrate that the scRNA-seq data are highly correlated with clinical outcome and hold potential for use as a companion diagnostic for MM and especially [primary refractory] MM patients," the authors wrote.