Identification of a CD138-Negative Therapy-Resistant Subpopulation in Multiple Myeloma with Vulnerability to Splicing Factor Inhibition

The molecular basis of therapy resistance in multiple myeloma remains poorly understood. In this study, we performed single-cell RNA sequencing coupled with VDJ-targeted sequencing of highly purified primary multiple myeloma cells from patient bone marrow. This approach uncovered cellular heterogeneity and phenotypic plasticity of multiple myeloma cells across a spectrum of CD138 expression, accompanied by drastic epigenetic alterations. Notably, therapy-resistant subpopulations were identified within a minor fraction of CD138- multiple myeloma cells, which were shown via CRISPR/Cas9 screening to be vulnerable to splicing pathway inhibition. Consistently, this fraction of CD138- multiple myeloma cells showed increased differential splicing associated with overexpression of SR protein family splicing factors. Among these splicing factors, RBM39 was overexpressed in therapy-resistant cells and involved in aberrant splicing. Both genetic and pharmacologic RBM39 inhibition exhibited a significant lethal effect on multiple myeloma cells. Collectively, our findings identify distinct therapy-resistant multiple myeloma subpopulations and highlight targeting the splicing pathway as a promising therapeutic strategy. SIGNIFICANCE: Single-cell RNA sequencing coupled with VDJ-targeted profiling identified distinct therapy-resistant subpopulations within the minor CD138- fraction of multiple myeloma cells. These subpopulations were characterized by increased differential splicing events associated with overexpression of splicing factors from the SR protein family, with CD138- cells showing selective vulnerability to pharmacologic targeting of the splicing factor RBM39. See related commentary by Maron and Abdel-Wahab, p. 535.