Identification a Novel Molecular Mechanism Underlying the Anti-CD38-Based Treatment Resistance in Multiple Myeloma Patients

Introduction: Anti-CD38 antibody (Daratumumab (DARA)/Isatuximab (ISA))-mediated therapy is now used in combination therapies to treat both newly diagnosed and relapsed/refractory multiple myeloma (MM). Despite a high extent and frequency of response to DARA/ISA containing therapy, MM patient relapse is still common. Several resistance mechanisms to DARA/ISA have been reported including MM cell clonal selection towards reduced CD38 expression; loss of MM cell surface CD38 upon the formation of a CD38-DARA complex leading to immunosuppression; and immunomodulatory effects after the DARA/ISA treatment via the downregulation of intracellular signaling pathways in bone marrow stromal cells. Given the widespread use of anti-CD38-directed treatment in MM, the identification of anti-CD38 based treatment resistance is an urgent clinical need.

Methods/results: To understand the underlying molecular mechanism of anti-CD38-based treatment resistance, we assessed post-transcriptional alterations on CD38 transcripts. We performed total and single-cell RNA sequencing of six samples from MM patients treated with DARA/ISA in combination with other MM drugs. Specifically, we examined mRNA splicing aberrations on the CD38 transcripts in 44 MM patient plasma cells (PCs). Four CD38 splice variants (CD38-Va, -Vb, -Vc, and Vd (CD38 SpVs)) were identified in CD138+PCs from DARA-resistant patient samples. These CD38-SpVs are the result of one or more cassette-exon splicing. Some of these splicing events cause in-frame shifts, which lead to the activation of a cryptic stop codon on the CD38 transcript and encode truncated proteins. The cryptic stop codon location at the specific proximity of the exon-intron junction, allowing these transcripts escaping degradation via non-sense mediated decay. In MM PCs, CD38 splice variant transcripts were overexpressed at different levels (2-4 fold) in various combinations with one or more novel splice variants and CD38 full-length (CD38-FL) transcripts. These splice variants were absent in MM PCs from BM aspirates from age-matched healthy donors.

Next, we evaluated the expression of the CD38 SpVs and FL transcripts in PCs from MM patients at the time they were sensitive to DARA/ISA treatment and when resistance developed. We identified CD38 splice variant transcripts that were selectively expressed MM PCs at the time of resistance to DARA/ISA treatment. We also identified CD38 splice variant transcripts that are overexpressed in DARA/ISA treatment-sensitive patient samples. Our findings were further validated in DARA resistant patient sample by single-cell RNA-seq analysis, which showed overexpression of CD38Vb in >80% of PCs. We identified splice site mutations that contribute to the expression of CD38 SpVs.

To evaluate whether CD38 aberrant splicing is associated with DARA resistance, we generated a KMS11 DARA-resistant cell line (KMS11 ^DARA-Res) and evaluated the expression of splice variants in these cells. This analysis demonstrated that KMS11^DARA-Res over-express CD38 SpVs.

To determine whether CD38 splice variants encode functional proteins, we cloned, sequenced, and stably expressed CD38 SpVs transcripts into H929 cells (H929-CD38 SpVs). CD38 SpVs expressions were evaluated by flow cytometry and live cell imaging. These analyses demonstrated that CD38 SpVs encode proteins. Importantly, CD38 splice variant protein folding analyses showed that splicing alterations abrogate DARA/ISA binding sites. Then, to evaluate the functional effects of aberrant splicing of CD38 in MM cells, we carried out DARA/ISA binding assays and evaluated cell killing with ADCC assays in H929- CD38SpV cells. Importantly, these analyses showed that both DARA/ISA binding and ADCC was lost in H929-CD38SpV cells.

Conclusions: We identified a novel molecular mechanism that underlies the resistance to anti-CD38-based treatment, both DARA and ISA. By this mechanism, CD38 cognate epitopes are removed from the cell surface of MM cells without an entire disposal of the target protein. The existence of such splicing-based mechanisms of resistance indicates that other antibody-based therapeutics or future CARs should be developed against CD38 epitopes that are created as a result of splicing. This approach may allow for selective targeting of MM PCs while spearing other CD38 expressing normal cells, enhancing cytotoxicity and improving therapeutic index.

Hatjiharissi:Janssen: Honoraria; Astra Zeneca: Honoraria; Lilly: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Research Funding; Genesis: Research Funding; Abbvie: Research Funding. Chu:Bristol Myers Squibb: Honoraria; Sanofi: Honoraria; Gilead: Honoraria; Janssen: Honoraria. Anderson:Pfizer: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Precision Biosciences: Membership on an entity's Board of Directors or advisory committees; Window: Membership on an entity's Board of Directors or advisory committees; Starton: Membership on an entity's Board of Directors or advisory committees; OncoPep: Other: Scientific founder ; C4 Therapeutics: Other: Scientific founder ; Raqia: Other: Scientific founder ; NextRNA: Other: Scientific founder ; Dynamic Cell Therapy: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Mana Therapeutics: Membership on an entity's Board of Directors or advisory committees.