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.

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.

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.

Successful inhibition of excitotoxic neuronal damage and microglial activation after delayed application of interleukin-1 receptor antagonist.

Interleukin (IL)-1 is an important mediator of neuronal demise and glial activation after acute central nervous system lesions and is antagonized by IL-1 receptor antagonist (IL-1RA). Here we determined the time window in which IL-1RA elicits neuroprotective effects in rat organotypic hippocampal slice cultures (OHSC). OHSC were lesioned with N-methyl-D-aspartate (NMDA) and treated with IL-1RA (100 ng/ml) at different time points postinjury or were left untreated. Damaged neurons, microglial cells, and astrocytes were labelled with NeuN, propidium iodide, isolectin B(4), or glial fibrillary acidic protein (GFAP), respectively, and were analyzed by confocal laser scanning microscopy. In lesioned OHSC, the most dramatic increase in microglial cell number occurred between 8 and 16 hr postinjury, and the maximal neuronal demise was found between 16 and 24 hr postinjury. The cellular source of IL-1beta was investigated by immunohistochemistry, and IL-1beta immunoreactivity was found in few microglial cells at 4 hr postinjury and in numerous microglial cells and astrocytes at 16 hr postinjury. In both glial populations, IL-1beta immunoreactivity peaked at 24 hr postinjury. IL-1RA treatment potently suppressed neuronal damage by 55% when initiated within the first 16 hr postinjury (P < 0.05), and IL-1RA treatment initiated at 24 hr postinjury resulted in weaker but still significant neuroprotection. IL-1RA treatment also reduced the number of microglial cells significantly when initiated within 36 hr postinjury (P < 0.05). In conclusion, IL-1RA exhibits significant neuroprotective effects in this in vitro model of excitotoxic injury even after delayed application.

[Influence of metabolic control on hemoglobin level in type 1 diabetic patients with nephropathy]. / Einfluss der Stoffwechseleinstellung auf den Hämoglobinspiegel bei Typ-1-Diabetikern mit Nephropathie.

BACKGROUND AND PURPOSE: Diabetic patients with nephropathy show a decline of hemoglobin even at a moderate degree of kidney dysfunction which may impair cardiovascular prognosis. As main reason a disturbed synthesis of erythropoietin (EPO) has been suggested, the pathogenesis, however, is unclear. The clinical significance of metabolic control for the hemoglobin and EPO levels was investigated. PATIENTS AND METHODS: The following parameters were determined in 185 type 1 diabetic patients: hemoglobin, HbA1c, calculated creatinine clearance, urinary albumin/creatinine ratio, lipids, high-sensitive C-reactive protein (hsCRP). Serum concentration of EPO was determined in 56 consecutive patients with renal dysfunction. RESULTS: Hemoglobin concentration decreased with declining renal function. Patients were stratified according to median HbA1c level (7.4%) in those with better (HbA1c < 7.4%) and worse metabolic control (HbA1c > 7.4%). In patients with kidney dysfunction, the group with better metabolic control showed higher hemoglobin concentrations than the group with worse metabolic control: 13.6 versus 12.6 g/dl at creatinine clearance < 60 ml/min (p = 0.02). Linear regression analysis revealed metabolic control aside from kidney function, gender, hsCRP and use of ACE inhibitiors as a significant influencing factor of hemoglobin concentration. In patients with renal dysfunction, EPO levels were higher in the group with better than worse metabolic control (13.0 vs. 9.8 U/l). CONCLUSION: The decline of hemoglobin in diabetic patients with renal dysfunction is mitigated in case of good metabolic control, possibly due to higher EPO concentrations. The results emphasize the clinical significance of a good metabolic control in diabetic patients with nephropathy.

De novo mutations in HCN1 cause early infantile epileptic encephalopathy.

Hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels contribute to cationic Ih current in neurons and regulate the excitability of neuronal networks. Studies in rat models have shown that the Hcn1 gene has a key role in epilepsy, but clinical evidence implicating HCN1 mutations in human epilepsy is lacking. We carried out exome sequencing for parent-offspring trios with fever-sensitive, intractable epileptic encephalopathy, leading to the discovery of two de novo missense HCN1 mutations. Screening of follow-up cohorts comprising 157 cases in total identified 4 additional amino acid substitutions. Patch-clamp recordings of Ih currents in cells expressing wild-type or mutant human HCN1 channels showed that the mutations had striking but divergent effects on homomeric channels. Individuals with mutations had clinical features resembling those of Dravet syndrome with progression toward atypical absences, intellectual disability and autistic traits. These findings provide clear evidence that de novo HCN1 point mutations cause a recognizable early-onset epileptic encephalopathy in humans.

Interleukin-1beta exacerbates and interleukin-1 receptor antagonist attenuates neuronal injury and microglial activation after excitotoxic damage in organotypic hippocampal slice cultures.

The effects of interleukin (IL)-1beta and IL-1 receptor antagonist (IL-1ra) on neurons and microglial cells were investigated in organotypic hippocampal slice cultures (OHSCs). OHSCs obtained from rats were excitotoxically lesioned after 6 days in vitro by application of N-methyl-D-aspartate (NMDA) and treated with IL-1beta (6 ng/mL) or IL-1ra (40, 100 or 500 ng/mL) for up to 10 days. OHSCs were then analysed by bright field microscopy after hematoxylin staining and confocal laser scanning microscopy after labeling of damaged neurons with propidium iodide (PI) and fluorescent staining of microglial cells. The specificity of PI labeling of damaged neurons was validated by triple staining with neuronal and glial markers and it was observed that PI accumulated in damaged neurons only but not in microglial cells or astrocytes. Treatment of unlesioned OHSCs with IL-1beta did not induce neuronal damage but caused an increase in the number of microglial cells. NMDA lesioning alone resulted in a massive increase in the number of microglial cells and degenerating neurons. Treatment of NMDA-lesioned OHSCs with IL-1beta exacerbated neuronal cell death and further enhanced microglial cell numbers. Treatment of NMDA-lesioned cultures with IL-1ra significantly attenuated NMDA-induced neuronal damage and reduced the number of microglial cells, whereas application of IL-1ra in unlesioned OHSCs did not induce significant changes in either cell population. Our findings indicate that: (i) IL-1beta directly affects the central nervous system and acts independently of infiltrating hematogenous cells; (ii) IL-1beta induces microglial activation but is not neurotoxic per se; (iii) IL-1beta enhances excitotoxic neuronal damage and microglial activation and (iv) IL-1ra, even when applied for only 4 h, reduces neuronal cell death and the number of microglial cells after excitotoxic damage.