Resolving therapy resistance mechanisms in multiple myeloma by multiomics subclone analysis.

Intratumor heterogeneity as a clinical challenge becomes most evident after several treatment lines, when multidrug-resistant subclones accumulate. To address this challenge, the characterization of resistance mechanisms at the subclonal level is key to identify common vulnerabilities. In this study, we integrate whole-genome sequencing, single-cell (sc) transcriptomics (scRNA sequencing), and chromatin accessibility (scATAC sequencing) together with mitochondrial DNA mutations to define subclonal architecture and evolution for longitudinal samples from 15 patients with relapsed or refractory multiple myeloma. We assess transcriptomic and epigenomic changes to resolve the multifactorial nature of therapy resistance and relate it to the parallel occurrence of different mechanisms: (1) preexisting epigenetic profiles of subclones associated with survival advantages, (2) converging phenotypic adaptation of genetically distinct subclones, and (3) subclone-specific interactions of myeloma and bone marrow microenvironment cells. Our study showcases how an integrative multiomics analysis can be applied to track and characterize distinct multidrug-resistant subclones over time for the identification of molecular targets against them.

Changes in T-cell subsets, preexisting cytopenias and hyperferritinaemia correlate with cytopenias after BCMA targeted CAR T-cell therapy in relapsed/refractory multiple myeloma: Results from a prospective comprehensive biomarker study.

Biomarkers for cytopenias following CAR T-cell treatment in relapsed/refractory (RR) multiple myeloma (MM) are not completely defined. We prospectively analysed 275 sequential peripheral blood (PB) samples from 58 RRMM patients treated with BCMA-targeted CAR T cells, and then divided them into three groups: (i) baseline (before leukapheresis), (ii) ≤day+30, and (iii) >day+30 after CAR T-cell therapy. We evaluated laboratory data and performed flow cytometry to determine the (CAR) T-cell subsets. Baseline hyperferritinaemia was a risk factor for long-lasting grade ≥3 anaemia (r = 0.47, p < 0.001) and thrombocytopenia (r = 0.44, p = 0.002) after CAR T-cell therapy. Low baseline haemoglobin (Hb) and PLT were associated with long-lasting grade ≥3 anaemia (r = -0.56, p < 0.001) and thrombocytopenia (r = -0.44, p = 0.002) respectively. We observed dynamics of CAR-negative T-cell subsets following CAR T-cell infusion. In the late phase after CAR T-cell therapy (>day+30), CD4Tn frequency correlated with anaemia (r = 0.41, p = 0.0014) and lymphocytopenia was related to frequencies of CD8+ T cells (r = 0.72, p < 0.001) and CD8Teff (r = 0.64, p < 0.001). CD4Tcm frequency was correlated with leucocytopenia (r = -0.49, p < 0.001). In summary, preexisting cytopenias and hyperferritinaemia indicated long duration of grade ≥3 post-CAR T-cell cytopenias. Prolonged cytopenia may be related to immune remodelling with a shift in the CAR-negative T-cell subsets following CAR T-cell therapy.

Soluble B-cell maturation antigen in lacrimal fluid as a potential biomarker and mediator of keratopathy in multiple myeloma.

Belantamab mafodotin (belantamab) is a first-in-class anti-BCMA antibody-drug conjugate approved for the treatment of triple-class refractory multiple myeloma. It provides a unique therapeutic option for patients ineligible for CAR-T and bispecific antibody therapy, and/or patients progressing on anti-CD38 treatment where CAR-T and bispecifics might be kept in reserve. Wider use of the drug can be challenged by its distinct ocular side effect profile, including corneal microcysts and keratopathy. While dose reduction has been the most effective way to reduce these toxicities, the underlying mechanism of this BCMA off-target effect remains to be characterized. In this study, we provide the first evidence for soluble BCMA (sBCMA) in lacrimal fluid and report on its correlation with tumor burden in myeloma patients. We confirm that corneal cells do not express BCMA, and show that sBCMA-belantamab complexes may rather be internalized by corneal epithelial cells through receptor-ligand independent pinocytosis. Using an hTcEpi corneal cell-line model, we show that the pinocytosis inhibitor EIPA significantly reduces belantamab-specific cell killing. As a proof of concept, we provide detailed patient profiles demonstrating that, after belantamab-induced cell killing, sBCMA is released into circulation, followed by a delayed increase of sBCMA in the tear fluid and subsequent onset of keratopathy. Based on the proposed mechanism, pinocytosis-induced keratopathy can be prevented by lowering the entry of sBCMA into the lacrimal fluid. Future therapeutic concepts may therefore consist of belantamab-free debulking therapy prior to belantamab consolidation and/or concomitant use of gamma-secretase inhibition as currently evaluated for belantamab and nirogacestat in ongoing studies.

Cereblon enhancer methylation and IMiD resistance in multiple myeloma.

Cereblon is the direct binding target of the immunomodulatory drugs (IMiDs) that are commonly used to treat multiple myeloma (MM), the second most frequent hematologic malignancy. Patients respond well to initial treatment with IMiDs, but virtually all patients develop drug resistance over time, and the underlying mechanisms are poorly understood. We identified an as yet undescribed DNA hypermethylation in an active intronic CRBN enhancer. Differential hypermethylation in this region was found to be increased in healthy plasma cells, but was more pronounced in IMiD-refractory MM. Methylation significantly correlated with decreased CRBN expression levels. DNA methyltransferase inhibitor (DNTMi) in vitro experiments induced CRBN enhancer demethylation, and sensitizing effects on lenalidomide treatment were observed in 2 MM cell lines. Thus, we provide first evidence that aberrant CRBN DNA methylation is a novel mechanism of IMiD resistance in MM and may predict IMiD response prior to treatment.

High-dose carfilzomib achieves superior anti-tumor activity over low-dose and recaptures response in relapsed/refractory multiple myeloma resistant to lowdose carfilzomib by co-inhibiting the ß2 and ß1 subunits of the proteasome complex.

Optimal carfilzomib dosing is a matter of debate. We analyzed the inhibition profiles of proteolytic proteasome subunits ß5, ß2 and ß1 after low-dose (20/27 mg/m2) versus high-dose (≥36 mg/m2) carfilzomib in 103 pairs of peripheral blood mononuclear cells from patients with relapsed/refractory (RR) multiple myeloma (MM). ß5 activity was inhibited (median inhibition >50%) in vivo by 20 mg/m2, whereas ß2 and ß1 were co-inhibited only by 36 and 56 mg/m2, respectively. Coinhibition of ß2 (P=0.0001) and ß1 activity (P=0.0005) differed significantly between high-dose and low-dose carfilzomib. Subsequently, high-dose carfilzomib showed significantly more effective proteasome inhibition than low-dose carfilzomib in vivo (P=0.0003). We investigated the clinical data of 114 patients treated with carfilzomib combinations. High-dose carfilzomib demonstrated a higher overall response rate (P=0.03) and longer progression-free survival (PFS) (P=0.007) than low-dose carfilzomib. Therefore, we escalated the carfilzomib dose to ≥36 mg/m2 in 16 patients who progressed during low-dose carfilzomib-containing therapies. High-dose carfilzomib recaptured response (≥ partial remission) in nine (56%) patients with a median PFS of 4.4 months. Altogether, we provide the first in vivo evidence in RRMM patients that the molecular activity of high-dose carfilzomib differs from that of low-dose carfilzomib by coinhibition of ß2 and ß1 proteasome subunits and, consequently, high-dose carfilzomib achieves a superior anti-MM effect than low-dose carfilzomib and recaptures the response in RRMM resistant to low-dose carfilzomib. The optimal carfilzomib dose should be ≥36 mg/m2 to reach a sufficient anti-tumor activity, while the balance between efficacy and tolerability should be considered in each patient.

Characterization of Mercury-Resistant Rhizobacteria for Plant Growth Promotion: An In Vitro and In Silico Approach.

In this study, a total 30 rhizobacterial isolates were screened out based on resistance against different concentrations of mercuric chloride (HgCl2), growth on nitrogen-free mannitol (NFM) and production of indole-3-acetic acid (IAA). The biochemical and plant growth promoting characterization of selected isolates was performed by different biochemical tests. Out of 30, six isolates, UM-3, AZ-5, UM-7, UM-11, UM-26, and UM-28 showed resistance at 30 µg/ml HgCl2, pronounced growth on NFM and high production of IAA as 18.6, 16.7, 16, 18.7, 14, and 16 µg/ml, respectively (P < 0.05). The 16S rDNA ribotyping and phylogenetic analysis of selected bacterial isolates were performed and characterized as Exiguobacterium sp. UM-3 (KJ736011), Bacillus thuringiensis AZ-5 (KJ675627), Bacillus subtilis UM-7 (KJ736013), Enterobacter cloacae UM-11 (KJ736014), Pseudomonas aeruginosa UM-26 (KJ736016), P. aeruginosa UM-28 (KJ736017) and Bacillus pumilus UM-16 (KJ736015) used as negative control. B. thuringiensis AZ-5 showed high resistance against 30 µg/ml of HgCl2 due to the presence of merB gene. The structural determination of MerB protein was carried out using bioinformatics tools, i.e., Protparam, Pfam, InterProScan, STRING, Jpred4, PSIPRED, I-TASSER, COACH server and ERRAT. These tools predicted the structural based functional homology of MerB protein (organomercuric lyase) in association with MerA (mercuric reductase) in bacterial Hg-detoxification system.

CIC Mutation as a Molecular Mechanism of Acquired Resistance to Combined BRAF-MEK Inhibition in Extramedullary Multiple Myeloma with Central Nervous System Involvement.

Combined MEK-BRAF inhibition is a well-established treatment strategy in BRAF-mutated cancer, most prominently in malignant melanoma with durable responses being achieved through this targeted therapy. However, a subset of patients face primary unresponsiveness despite presence of the activating mutation at position V600E, and others acquire resistance under treatment. Underlying resistance mechanisms are largely unknown, and diagnostic tests to predict tumor response to BRAF-MEK inhibitor treatment are unavailable. Multiple myeloma represents the second most common hematologic malignancy, and point mutations in BRAF are detectable in about 10% of patients. Targeted inhibition has been successfully applied, with mixed responses observed in a substantial subset of patients mirroring the widespread spatial heterogeneity in this genomically complex disease. Central nervous system (CNS) involvement is an extremely rare, extramedullary form of multiple myeloma that can be diagnosed in less than 1% of patients. It is considered an ultimate high-risk feature, associated with unfavorable cytogenetics, and, even with intense treatment applied, survival is short, reaching less than 12 months in most cases. Here we not only describe the first patient with an extramedullary CNS relapse responding to targeted dabrafenib and trametinib treatment, we furthermore provide evidence that a point mutation within the capicua transcriptional repressor (CIC) gene mediated the acquired resistance in this patient. KEY POINTS: BRAF mutations constitute an attractive druggable target in multiple myeloma. This is the first genomic dissection of the central nervous system involvement in a multiple myeloma patient harboring a druggable BRAFV600E mutation. Deep genomic characterization of the extramedullary lesion prompted a personalized therapeutic approach. Acquisition of CIC mutation confers a mechanism of BRAF-MEK inhibitor drug resistance in multiple myeloma. The in silico interrogation of the CoMMpass clinical study revealed 10 patients with somatic mutations of CIC and its downregulation at gene expression level in multiple myeloma. CIC gene silencing decreases the sensitivity of multiple myeloma cells to BRAF-MEK inhibition in vitro. The correlation between CIC downregulation and ETV4/5 nuclear factor expression in multiple myeloma BRAF-mutant cells is shown for the first time. CIC mutation, its downregulation, and the related downstream effect on MMP24 support disseminative potential providing new clues in the extramedullary biology definition.

Clonal competition assays identify fitness signatures in cancer progression and resistance in multiple myeloma.

Multiple myeloma (MM) is a genetically heterogeneous disease and the management of relapses is one of the biggest clinical challenges. TP53 alterations are established high-risk markers and are included in the current disease staging criteria. KRAS is the most frequently mutated gene affecting around 20% of MM patients. Applying Clonal Competition Assays (CCA) by co-culturing color-labeled genetically modified cell models, we recently showed that mono- and biallelic alterations in TP53 transmit a fitness advantage to the cells. Here, we report a similar dynamic for two mutations in KRAS (G12A and A146T), providing a biological rationale for the high frequency of KRAS and TP53 alterations at MM relapse. Resistance mutations, on the other hand, did not endow MM cells with a general fitness advantage but rather presented a disadvantage compared to the wild-type. CUL4B KO and IKZF1 A152T transmit resistance against immunomodulatory agents, PSMB5 A20T to proteasome inhibition. However, MM cells harboring such lesions only outcompete the culture in the presence of the respective drug. To better prevent the selection of clones with the potential of inducing relapse, these results argue in favor of treatment-free breaks or a switch of the drug class given as maintenance therapy. In summary, the fitness benefit of TP53 and KRAS mutations was not treatment-related, unlike patient-derived drug resistance alterations that may only induce an advantage under treatment. CCAs are suitable models for the study of clonal evolution and competitive (dis)advantages conveyed by a specific genetic lesion of interest, and their dependence on external factors such as the treatment.

Genetic Alterations in Members of the Proteasome 26S Subunit, AAA-ATPase (PSMC) Gene Family in the Light of Proteasome Inhibitor Resistance in Multiple Myeloma.

For the treatment of Multiple Myeloma, proteasome inhibitors are highly efficient and widely used, but resistance is a major obstacle to successful therapy. Several underlying mechanisms have been proposed but were only reported for a minority of resistant patients. The proteasome is a large and complex machinery. Here, we focus on the AAA ATPases of the 19S proteasome regulator (PSMC1-6) and their implication in PI resistance. As an example of cancer evolution and the acquisition of resistance, we conducted an in-depth analysis of an index patient by applying FISH, WES, and immunoglobulin-rearrangement sequencing in serial samples, starting from MGUS to newly diagnosed Multiple Myeloma to a PI-resistant relapse. The WES analysis uncovered an acquired PSMC2 Y429S mutation at the relapse after intensive bortezomib-containing therapy, which was functionally confirmed to mediate PI resistance. A meta-analysis comprising 1499 newly diagnosed and 447 progressed patients revealed a total of 36 SNVs over all six PSMC genes that were structurally accumulated in regulatory sites for activity such as the ADP/ATP binding pocket. Other alterations impact the interaction between different PSMC subunits or the intrinsic conformation of an individual subunit, consequently affecting the folding and function of the complex. Interestingly, several mutations were clustered in the central channel of the ATPase ring, where the unfolded substrates enter the 20S core. Our results indicate that PSMC SNVs play a role in PI resistance in MM.

Bortezomib induced peripheral neuropathy and single nucleotide polymorphisms in PKNOX1.

We analyzed single nucleotide polymorphisms (SNPs) in PKNOX1 (rs2839629) and in the intergenic region between PKNOX1 and CBS (rs915854) by Sanger sequencing in 88 patients with multiple myeloma treated with bortezomib. All patients (n = 13) harboring a homozygous mutation in PKNOX1 (rs2839629) also had a homozygous mutated rs915854 genotype. Homozygous mutated genotypes of rs2839629 and rs915854 were significantly enriched in patients with painful peripheral neuropathy (PNP) (P < 0.0001), and homozygous mutated rs2839629 genotype was significantly enriched in patients with pain compared to patients with no pain (P = 0.04). In summary, both SNPs rs2839629 and/or rs915854 may be potential biomarkers predicting an increased risk to develop painful PNP under bortezomib.

Single-Nucleotide Variants and Epimutations Induce Proteasome Inhibitor Resistance in Multiple Myeloma.

PURPOSE: Proteasome inhibitors (PI) are the backbone of various treatment regimens in multiple myeloma. We recently described the first in-patient point mutations affecting the 20S subunit PSMB5 underlying PI resistance. Notably, in vivo, the incidence of mutations in PSMB5 and other proteasome encoding genes is too low to explain the development of resistance in most of the affected patients. Thus, additional genetic and epigenetic alterations need to be explored. EXPERIMENTAL DESIGN: We performed DNA methylation profiling by Deep Bisulfite Sequencing in PSMB5, PSMC2, PSMC5, PSMC6, PSMD1, and PSMD5, a subset of proteasome subunits that have hitherto been associated with PI resistance, recruited from our own previous research, the literature, or a meta-analysis on the frequency of somatic mutations. Methylation was followed up on gene expression level and by dual-luciferase reporter assay. The KMS11 cell line served as a model to functionally test the impact of demethylating agents. RESULTS: We identified PSMD5 promoter hypermethylation and subsequent epigenetic gene silencing in 24% of PI refractory patients. Hypermethylation correlated with decreased expression and the regulatory impact of this region was functionally confirmed. In contrast, patients with newly diagnosed multiple myeloma, along with peripheral blood mononuclear cells and CD138+ plasma cells from healthy donors, generally show unmethylated profiles. CONCLUSIONS: Under the selective pressure of PI treatment, multiple myeloma cells acquire methylation of the PSMD5 promoter silencing the PSMD5 gene expression. PSMD5 acts as a key orchestrator of proteasome assembly and its downregulation was described to increase the cell's proteolytic capacity. PSMD5 hypermethylation, therefore, represents a novel mechanism of PI tolerance in multiple myeloma.

Impaired FADD/BID signaling mediates cross-resistance to immunotherapy in Multiple Myeloma.

The treatment landscape in multiple myeloma (MM) is shifting from genotoxic drugs to immunotherapies. Monoclonal antibodies, immunoconjugates, T-cell engaging antibodies and CART cells have been incorporated into routine treatment algorithms, resulting in improved response rates. Nevertheless, patients continue to relapse and the underlying mechanisms of resistance remain poorly understood. While Impaired death receptor signaling has been reported to mediate resistance to CART in acute lymphoblastic leukemia, this mechanism yet remains to be elucidated in context of novel immunotherapies for MM. Here, we describe impaired death receptor signaling as a novel mechanism of resistance to T-cell mediated immunotherapies in MM. This resistance seems exclusive to novel immunotherapies while sensitivity to conventional anti-tumor therapies being preserved in vitro. As a proof of concept, we present a confirmatory clinical case indicating that the FADD/BID axis is required for meaningful responses to novel immunotherapies thus we report impaired death receptor signaling as a novel resistance mechanism to T-cell mediated immunotherapy in MM.

CXCR4 expression of multiple myeloma as a dynamic process: influence of therapeutic agents.

Chemokine receptors represent novel targets for treatment of multiple myeloma (MM). However, CXCR4 expression appears to be highly dynamic. This in vitro study investigated the impact of commonly used anti-myeloma agents on CXCR4 expression. Established human myeloma cell lines as well as patient-derived CD138+ plasma cells were exposed to antineoplastic drugs. Cells were analyzed for CXCR4 expression by flow cytometry and direct stochastic optical reconstruction microscopy (dSTORM). In addition, cellular uptake of 68Ga-Pentixafor, a PET radiotracer for noninvasive assessment of CXCR4 expression in vivo, was assessed. CXCR4 expression was highly variable and turned out to be substance, dose and time dependent. Treatment with bortezomib was associated with reduced expression, while dexamethasone and doxorubicin significantly increased expression of CXCR4. Combination of these compounds further increased CXCR4 expression. In conclusion, drugs or combination of drugs can induce CXCR4 expression in myeloma cells. Hence, pretreatment may impact on response to CXCR4-based therapies.

Assessment of TP53 lesions for p53 system functionality and drug resistance in multiple myeloma using an isogenic cell line model.

Recent advances in molecular diagnostics have shown that lesions affecting both copies of the gene for tumor suppressor protein 53 (TP53) count among the most powerful predictors for high-risk disease in multiple myeloma (MM). However, the functional relevance and potential therapeutic implications of single hits to TP53 remain less well understood. Here, we have for the first time approximated the different constellations of mono- and bi-allelic TP53 lesions observed in MM patients within the frame of a single MM cell line model and assessed their potential to disrupt p53 system functionality and to impart drug resistance. Both types of common first hit: point mutation with expression of mutant p53 protein or complete loss of contribution from one of two wildtype alleles strongly impaired p53 system functionality and increased resistance to melphalan. Second hits abolished remaining p53 activity and increased resistance to genotoxic drugs even further. These results fit well with the clinical drive to TP53 single- and double-hit disease in MM patients, provide a rationale for the most commonly observed double-hit constellation (del17p+ TP53 point mutation), and underscore the potential increases in MM cell malignancy associated with any type of initial TP53 lesion.