Multiples Myelom IV
V595 In Richtung einer Progressions- und Entzündungssignatur der aberranten Notch-Signalgebung im Myelom
Daniela Simone Maichl, Würzburg, D
Aberrant Notch signaling is a key regulator of onset, progression, and drug resistance in myeloma (MM). Molecular and biological functions specifically attributed to Notch 1 (N1) and Notch 2 (N2) receptor signaling in MM are mainly unknown. In our study shRNA-based N1 and N2 knockdown (KD) in MM.1S and RPMI 8226 MM cells decreased viability and increased drug sensitivity to lenalidomide, bortezomib, and melphalan. To identify N1 and N2 specific gene expression patterns accounting for the tumor biologic effects, we performed genome-wide high-throughput RNA sequencing (RNAseq) of MM cells. RNAseq data were analyzed using the EdgeR software package. Bioinformatic analysis comprised a comparison of regulated genes in all conditions (N1 KD vs. N2 KD vs. control; padj < 0.01), Gene Ontology (GO) analysis (padj < 0.15), and gene set enrichment analysis (GSEA; FDR q-value < 0.25). We showed that N1 and N2 signaling contribute to a progression signature in MM. Expression of the cytokines BAFF, APRIL, CXCL9, and CXCL12 known to drive MM growth, progression, and survival were downregulated after Notch depletion. Intriguingly, N1 and N2 KD resulted in suppression of the newly identified E3 ligase tripartite motif-containing protein 21 which is implemented in the destabilization of the tumor suppressor p53 and the ubiquitination and enhanced degradation of p27 mediating resistance to proteasome inhibitor bortezomib in MM. Furthermore, KD of both receptors strongly downregulated expression of seven members of the JAK-STAT pathway including STAT3, which has been shown to have an adverse prognostic impact in MM and serves as a promising therapeutic target. MM progression is further accelerated through a characteristic inflammatory and immunosuppressive bone marrow milieu. GSEA results revealed significant transcriptional changes of pro inflammatory cytokines like CCL2, CCL3, and IL-15, as well as immunosuppressive cytokines like IL-10 and APRIL through N1 and N2. Recently, a bone-resident cell-like phenotype has been described in MM cells in which bone-related genes such as osteopontin, cathepsin K, and RANK are expressed that mimic bone marrow resident cells. Expression of this particular set of genes was significantly suppressed in N1 KD MM cells highlighting its role in supporting MM progression in bone. Our findings provide new insights into the multifaceted function of N1 and N2 signaling and emphasize the crucial role of the Notch pathway as a rational target in MM.
V613 Identifikation von CDK6 als therapeutisches Target beim rezidivierten Multiplen Myelom durch Proteom-Analysen
Christian Könecke, Hannover, D
Introduction: The immunomodulatory drugs (IMiDs) lenalidomide and pomalidomide are highly effective treatments for multiple myeloma but acquired resistance is a major clinical challenge. Comprehensive sequencing analyses of resistant patients revealed resistance-causing genetic alterations in the CRBN-CRL4 E3 ligase, the primary target of IMiDs, and other genes only in a small subset of patients. This implies that non-genetic resistance mechanisms for IMiDs exist.
Methods: In order to identify deregulated protein and RNA expression in relapsed multiple myeloma, we performed global quantitative tandem mass tag (TMT)-based proteomics and RNA sequencing in paired pre-treatment and relapse samples from five patients treated with lenalidomide. Functional analyses including genetic and pharmacologic perturbations were performed in multiple myeloma cell lines.
Results: Comparative proteomic analyses revealed proteins highly upregulated in relapsed/ resistant multiple myeloma, including the cell cycle regulator CDK6, as well as proteins previously found implicated in drug resistant multiple myeloma like RRM1 and TRIP13. None of these proteins were found significantly deregulated on the RNA level. Overexpression of CDK6 and RRM1 in multiple myeloma cell lines reduced sensitivity to lenalidomide and pomalidomide but not to other drugs. Further experiments identified CDK6 as master regulator of a relapse-associated protein expression signature, including RRM1 and TRIP13, that is reversible by inactivating CDK6. While CDK6 inhibition alone has only modest effects on multiple myeloma cell lines, it is highly synergistic with lenalidomide and pomalidomide in an Rb1-independent fashion. Furthermore, pomalidomide-based proteolysis targeting chimeras (PROTACs) targeting CDK6 and IKZF1/3 for degradation were highly active even in IMiD-insensitive multiple myeloma cell lines.
Conclusions: Proteomic profiling of primary multiple myeloma cells identifies a CDK6-governed program as a non-genetic resistance mechanism. Pharmacologic targeting of CDK6 may be a promising strategy to overcome IMiD resistance in multiple myeloma.
V668 Multiples Myelom und klinische Studien: Limitierte Teilnahme aufgrund restriktiver Ein- und Ausschlußkriterien sowie Erwartungen der Patienten
Amelie Boquoi, Düsseldorf, D
Randomized controlled trials (RCT) are the motor of pharmaceutical innovations. However, restrictive eligibility criteria as well as patient expectations often limit the proportion of patients treated within RCT. We provide a retrospective single-center analysis of 411 patients with multiple myeloma (MM) who were
treated at the University Hospital Düsseldorf in Germany from January 2014 to December 2016. Each patient was reviewed for the possibility of participating in a therapy study based on the recruiting period of the study and the inclusion and exclusion (I/E) criteria of 4 major and 2 additional international RCT.
The overall study participation of the analyzed population was 36%. 55% of 335 patients were eligible for first-line studies (LenaMain, GMMG-HD6) and 80% of these were included (45% of all). Study eligibility for relapsed/refractory patients varied between 22-51% (GMMG-Relapse, Castor, Tourmaline, and Admyre). Only 15-33% of these (5-12% of all) consented to enrollment.
For the majority of cases, only one I/E criteria rendered patients ineligible. Study-specific criteria (prior therapies, refractory disease) and second primary malignancy were the most common I/E criteria followed by internal, neurologic and infectious disease.
In summary, this single-center analysis shows that only a subset of the patient population had access to trials conducted and that the willingness to participate decreased during the course of the disease. At diagnosis, trial participation is usually high because efficacy is prioritized by myeloma patients, while at relapse quality-of-life and convenience were considered more important.
V521 Idecabtagen Vicleucel (Ide-Cel, bb2121), eine gegen BCMA-gerichtete CAR-T-Zelltherapie zur Behandlung von Patienten mit rezidiviertem und refraktärem Multiplen Myelom: Update der KarMMa-Studie
Hermann Einsele, Würzburg, D
Introduction: Patients (pts) with relapsed and refractory multiple myeloma (RRMM) previously exposed to IMiDs, proteasome inhibitors (PIs), and anti-CD38 monoclonal antibodies (mAbs) have poor outcomes with subsequent treatments. In the pivotal KarMMa trial, ide-cel, a BCMA-directed CAR T-cell therapy, showed frequent, deep, and durable responses in heavily pretreated pts with RRMM (Munshi et al. N Engl J Med. 2021;384:705-716). This analysis reports updated efficacy and safety data from the phase 2 KarMMa trial (NCT03361748).
Methods: Eligible pts had received ≥ 3 prior regimens (including an IMiD, a PI, and an anti-CD38 mAb) and were refractory to the last regimen. Following lymphodepletion, pts received 150─450 × 106 CAR+ T cells (target dose levels). The primary endpoint was overall response rate (ORR). Secondary endpoints included complete response (CR) rate, duration of response (DOR), progression-free survival (PFS), overall survival (OS), and safety.
Results: Of the 140 pts enrolled in KarMMa, 128 received ide-cel. Data are presented for the treated pts, who had a median age of 61 years and a median of 6 (range, 3-16) prior regimens; 84% were triple- class refractory, most pts (88%) had received bridging therapy. At the data cutoff (April 7, 2020), the median follow-up was 15.4 months. The ORR was 73%, and the median PFS was 8.8 months; both increased with higher dose (Table). At the highest target dose (450 × 106 CAR+ T cells), the ORR was 81%, CR rate was 39%, and median PFS increased to 12.2 months with longer follow-up. Responses were observed in all subgroups, including difficult-to-treat subsets (eg, high tumor burden [ORR, 71%], extramedullary disease [70%], and R-ISS stage III disease [48%]). OS continues to mature, and the median has not been reached (Figure); the 15-month event-free rate for OS was 71%. The most common any-grade toxicities were cytopenias (97%) and cytokine release syndrome (CRS; 84%, majority grade 1/2).
Conclusions: Updated results of the KarMMa trial continue to demonstrate deep, durable responses with ide-cel, supporting a favorable clinical benefit-risk profile in heavily pretreated, triple class-exposed pts with RRMM.
V675 Detaillierte Charakterisierung der OTUD6B-LIN28B-Achse als wichtiger Treiber im Multiplen Myelom
Introduction: Multiple myeloma (MM) is the second most common hematological malignancy that remains incurable, thus demanding for new therapeutic approaches. The substantial responsiveness of MM patients to proteasomal inhibitors hints towards a central role of the ubiquitin proteasome system (UPS) in MM. Therefore, investigating components of the UPS may lead to the identification of new druggable vulnerabilities in this disease. Deubiquitylases (DUBs) are particularly attractive, as they can readily be targeted by small molecule inhibitors.
Methods: A pooled CRISPR/Cas9 screen targeting all human DUBs was performed in MM cells to identify new vulnerabilities. For validated candidates, phenotypical analysis regarding proliferation and cell cycle progression was performed, as well affinity and non-affinity mass spectrometry (MS)-based screens to identify substrates. Binding and deubiquitylation of a substrate candidate were confirmed by immunoprecipitation, in vivo and in vitro ubiquitylation experiments. DUB activity assays and cycloheximide experiments were conducted at different cell cycle stages. Xenograft experiments were conducted with OTUD6B or LIN28B depleted MM cells, as well as RNA-Seq analyses. mRNA levels of MYC were analyzed by qPCR in MM cell lines and patient derived samples.
Results: In a pooled CRISPR/Cas9 screen for dependencies among the family of DUBs in MM, we identified OTUD6B as the most significant hit and unraveled its cell biological function as a central mediator of cell cycle progression at the G1/S transition. The RNA binding protein LIN28B was identified as the bona fide deubiquitylation substrate of OTUD6B at G1/S, that binds to OTUD6B subsequent to phosphorylation. As a consequence, LIN28B becomes stabilized to mediate high expression of MYC that in turn promotes cell cycle progression. Analyses in large MM patient cohorts revealed a positive correlation between OTUD6B and MYC expression in primary patient samples as well as a correlation of high OTUD6B levels and adverse outcome.
Conclusions: Our studies identify the OTUD6B-LIN28B axis as a potent driver of MM that activates G1/S specific MYC expression and specify this nexus as a promising vulnerability in MM.