The WERA cancer center matrix: Strategic management of patient access to precision oncology in a large and mostly rural area of Germany.

PURPOSE: Providing patient access to precision oncology (PO) is a major challenge of clinical oncologists. Here, we provide an easily transferable model from strategic management science to assess the outreach of a cancer center. METHODS: As members of the German WERA alliance, the cancer centers in Würzburg, Erlangen, Regensburg and Augsburg merged care data regarding their geographical impact. Specifically, we examined the provenance of patients from WERA´s molecular tumor boards (MTBs) between 2020 and 2022 (n = 2243). As second dimension, we added the provenance of patients receiving general cancer care by WERA. Clustering our catchment area along these two dimensions set up a four-quadrant matrix consisting of postal code areas with referrals towards WERA. These areas were re-identified on a map of the Federal State of Bavaria. RESULTS: The WERA matrix overlooked an active screening area of 821 postal code areas - representing about 50 % of Bavaria´s spatial expansion and more than six million inhabitants. The WERA matrix identified regions successfully connected to our outreach structures in terms of subsidiarity - with general cancer care mainly performed locally but PO performed in collaboration with WERA. We also detected postal code areas with a potential PO backlog - characterized by high levels of cancer care performed by WERA and low levels or no MTB representation. CONCLUSIONS: The WERA matrix provided a transparent portfolio of postal code areas, which helped assessing the geographical impact of our PO program. We believe that its intuitive principle can easily be transferred to other cancer centers.

Ancistrocladinium A Induces Apoptosis in Proteasome Inhibitor-Resistant Multiple Myeloma Cells: A Promising Therapeutic Agent Candidate.

The N,C-coupled naphthylisoquinoline alkaloid ancistrocladinium A belongs to a novel class of natural products with potent antiprotozoal activity. Its effects on tumor cells, however, have not yet been explored. We demonstrate the antitumor activity of ancistrocladinium A in multiple myeloma (MM), a yet incurable blood cancer that represents a model disease for adaptation to proteotoxic stress. Viability assays showed a potent apoptosis-inducing effect of ancistrocladinium A in MM cell lines, including those with proteasome inhibitor (PI) resistance, and in primary MM cells, but not in non-malignant blood cells. Concomitant treatment with the PI carfilzomib or the histone deacetylase inhibitor panobinostat strongly enhanced the ancistrocladinium A-induced apoptosis. Mass spectrometry with biotinylated ancistrocladinium A revealed significant enrichment of RNA-splicing-associated proteins. Affected RNA-splicing-associated pathways included genes involved in proteotoxic stress response, such as PSMB5-associated genes and the heat shock proteins HSP90 and HSP70. Furthermore, we found strong induction of ATF4 and the ATM/H2AX pathway, both of which are critically involved in the integrated cellular response following proteotoxic and oxidative stress. Taken together, our data indicate that ancistrocladinium A targets cellular stress regulation in MM and improves the therapeutic response to PIs or overcomes PI resistance, and thus may represent a promising potential therapeutic agent.

In Silico, In Vitro, and In Vivo Investigations on Adapalene as Repurposed Third Generation Retinoid against Multiple Myeloma and Leukemia.

The majority of hematopoietic cancers in adults are incurable and exhibit unpredictable remitting-relapsing patterns in response to various therapies. The proto-oncogene c-MYC has been associated with tumorigenesis, especially in hematological neoplasms. Therefore, targeting c-MYC is crucial to find effective, novel treatments for blood malignancies. To date, there are no clinically approved c-MYC inhibitors. In this study, we virtually screened 1578 Food and Drug Administration (FDA)-approved drugs from the ZINC15 database against c-MYC. The top 117 compounds from PyRx-based screening with the best binding affinities to c-MYC were subjected to molecular docking studies with AutoDock 4.2.6. Retinoids consist of synthetic and natural vitamin A derivatives. All-trans-retinoic acid (ATRA) were highly effective in hematological malignancies. In this study, adapalene, a third-generation retinoid usually used to treat acne vulgaris, was selected as a potent c-MYC inhibitor as it robustly bound to c-MYC with a lowest binding energy (LBE) of -7.27 kcal/mol, a predicted inhibition constant (pKi) of 4.69 µM, and a dissociation constant (Kd value) of 3.05 µM. Thus, we examined its impact on multiple myeloma (MM) cells in vitro and evaluated its efficiency in vivo using a xenograft tumor zebrafish model. We demonstrated that adapalene exerted substantial cytotoxicity against a panel of nine MM and two leukemic cell lines, with AMO1 cells being the most susceptible one (IC50 = 1.76 ± 0.39 µM) and, hence, the focus of this work. Adapalene (0.5 × IC50, 1 × IC50, 2 × IC50) decreased c-MYC expression and transcriptional activity in AMO1 cells in a dose-dependent manner. An examination of the cell cycle revealed that adapalene halted the cells in the G2/M phase and increased the portion of cells in the sub-G0/G1 phase after 48 and 72 h, indicating that cells failed to initiate mitosis, and consequently, cell death was triggered. Adapalene also increased the number of p-H3(Ser10) positive AMO1 cells, which is a further proof of its ability to prevent mitotic exit. Confocal imaging demonstrated that adapalene destroyed the tubulin network of U2OS cells stably transfected with a cDNA coding for α-tubulin-GFP, refraining the migration of malignant cells. Furthermore, adapalene induced DNA damage in AMO1 cells. It also induced apoptosis and autophagy, as demonstrated by flow cytometry and western blotting. Finally, adapalene impeded tumor growth in a xenograft tumor zebrafish model. In summary, the discovery of the vitamin A derivative adapalene as a c-MYC inhibitor reveals its potential as an avant-garde treatment for MM.

[New forms of immunotherapy in uro-oncology : HLA-independent therapeutic approaches with bispecific antibodies and CAR T cells]. / Neue Formen der Immuntherapie für die Uroonkologie : HLA-unabhängige Therapieansätze mit bispezifischen Antikörpern und CAR-T-Zellen.

Immunotherapies using bispecific antibodies and chimeric antigen receptor (CAR) T cells do not depend on previous activation of T cells by the human leukocyte antigen (HLA) system. These HLA-independent approaches displayed groundbreaking clinical results in hematological malignancies-leading to drug approvals for diseases like acute lymphocytic leukemia (ALL), B-cell Non-Hodgkin's lymphoma and multiple myeloma. Currently, several phase I/II trials are investigating the transferability of these results to solid tumors-especially prostate cancer. Compared to established immune checkpoint blockade, bispecific antibodies and CAR T cells have novel and heterogenous side effects such as cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS). Treating these side effects and identifying suitable trial participants requires an interdisciplinary treatment approach.

Cynaropicrin disrupts tubulin and c-Myc-related signaling and induces parthanatos-type cell death in multiple myeloma.

The majority of blood malignancies is incurable and has unforeseeable remitting-relapsing paths in response to different treatments. Cynaropicrin, a natural sesquiterpene lactone from the edible parts of the artichoke plant, has gained increased attention as a chemotherapeutic agent. In this study, we investigated the effects of cynaropicrin against multiple myeloma (MM) cells in vitro and assessed its in vivo effectiveness in a xenograft tumor zebrafish model. We showed that cynaropicrin exerted potent cytotoxicity against a panel of nine MM cell lines and two leukemia cell lines with AMO1 being the most sensitive cell line (IC50 = 1.8 ± 0.3 µM). Cynaropicrin (0.8, 1.9, 3.6 µM) dose-dependently reduced c-Myc expression and transcriptional activity in AMO1 cells that was associated with significant downregulation of STAT3, AKT, and ERK1/2. Cell cycle analysis showed that cynaropicrin treatment arrested AMO1 cells in the G2M phase along with an increase in the sub-G0G1 phase after 24 h. With prolonged treatment times, cells accumulated more in the sub-G0G1 phase, implying cell death. Using confocal microscopy, we revealed that cynaropicrin disrupted the microtubule network in U2OS cells stably expressing α-tubulin-GFP. Furthermore, we revealed that cynaropicrin promoted DNA damage in AMO1 cells leading to PAR polymer production by PARP1 hyperactivation, resulting in AIF translocation from the mitochondria to the nucleus and subsequently to a novel form of cell death, parthanatos. Finally, we demonstrated that cynaropicrin (5, 10 µM) significantly reduced tumor growth in a T-cell acute lymphoblastic leukemia (T-ALL) xenograft zebrafish model. Taken together, these results demonstrate that cynaropicrin causes potent inhibition of hematopoietic tumor cells in vitro and in vivo.

A phase 2 clinical trial of combined BRAF/MEK inhibition for BRAFV600E-mutated multiple myeloma.

Activating BRAF mutations are found in a small subset of patients with newly diagnosed multiple myeloma, but prevalence increases in late-stage, refractory disease, and the mutations are associated with adverse outcome. This prospective single-arm, open-label, multicenter phase 2 trial assessed the efficacy and safety of combined BRAF/MEK inhibition, using encorafenib and binimetinib, in patients with relapsed/refractory multiple myeloma (RRMM) carrying a BRAFV600E mutation. Patients received 450 mg encorafenib once daily and binimetinib 45 mg twice daily. The primary end point was the overall response rate achieved within the first year after start of treatment according to International Myeloma Working Group criteria. Twelve RRMM patients with a median of 5 prior lines of therapy were enrolled. The overall response rate was 83.3%, with 10 patients achieving at least a partial response. The median progression-free survival was 5.6 months, and overall survival was 55% at 24 months. Emerging resistance to therapy was driven by RAS mutations and structural variants involving the BRAF locus. This is the first prospective clinical trial to demonstrate that combined BRAF/MEK inhibition is highly effective in patients with BRAFV600E-mutated RRMM, and it represents a successful targeted precision medicine approach in this disease. This trial was registered at www.clinicaltrials.gov as #NCT02834364.

Disruption of Lipid Raft Microdomains, Regulation of CD38, TP53, and MYC Signaling, and Induction of Apoptosis by Lomitapide in Multiple Myeloma Cells.

BACKGROUND/AIM: Multiple myeloma (MM) is characterized by accumulation of a malignant clone of plasma cells in the bone marrow. Curative treatments are not yet available. Therefore, we undertook a drug repurposing approach to identify possible candidates from a chemical library of 1,230 FDA-approved drugs by virtual drug screening. As a target, we have chosen the non-receptor Bruton's tyrosine kinase (BTK) which is one of the main regulators of the MM biomarker CD38. MATERIALS AND METHODS: In silico virtual screening was performed by using PyRx. Flow cytometry was applied for cell cycle and apoptosis analysis. Furthermore, protein and gene expression was determined by western blotting and microarray hybridization. Lipid raft staining was observed by confocal microscopy. RESULTS: The in silico identified lipid-lowering lomitapide presented with the strongest cytotoxicity among the top 10 drug candidates. This drug arrested the cell cycle in the G2/M phase and induced apoptosis in MM cells. Western blot analyses revealed that treatment with lomitapide induced cleavage of the apoptosis regulator PARP and reduced the expression of CD38, an integral part of lipid rafts. Using confocal microscopy, we further observed that lipid raft microdomain formation in MM cells was inhibited by lomitapide. In four MM cell lines (KMS-12-BM, NCI-H929, RPMI-8226, and MOLP-8) treated with lomitapide, microarray analyses showed not only that the expression of CD38 and BTK was down-regulated, but also that the tumor suppressor gene TP53 and the oncogene c-MYC were among the top deregulated genes. Further analysis of these data by Ingenuity pathway analysis (IPA) suggested that lomitapide interferes with the cross-talk of CD38 and BTK and apoptosis-regulating genes via TP53 and c-MYC. CONCLUSION: Lomitapide treatment led to disruption of lipid raft domains and induction of pro-apoptotic factors and might, therefore, be considered as a potential therapeutic agent in MM.

Results from a First-in-Human Phase I Study of Siremadlin (HDM201) in Patients with Advanced Wild-Type TP53 Solid Tumors and Acute Leukemia.

PURPOSE: This phase I, dose-escalation study investigated the recommended dose for expansion (RDE) of siremadlin, a p53-MDM2 inhibitor, in patients with wild-type TP53 advanced solid or hematologic cancers. PATIENTS AND METHODS: Initial dosing regimens were: 1A (day 1; 21-day cycle; dose 12.5-350 mg) and 2A (days 1-14; 28-day cycle; dose 1-20 mg). Alternative regimens included 1B (days 1 and 8; 28-day cycle) and 2C (days 1-7; 28-day cycle). The primary endpoint was incidence of dose-limiting toxicities (DLT) during cycle 1. RESULTS: Overall, 115 patients with solid tumors and 93 with hematologic malignancies received treatment. DLTs occurred in 8/92 patients with solid tumors and 10/53 patients with hematologic malignancies. In solid tumors, an RDE of 120 mg was defined in 1B. In hematologic tumors, RDEs were defined in 1A: 250 mg, 1B: 120 mg, and 2C: 45 mg. More patients with hematologic malignancies compared with solid tumors experienced grade 3/4 treatment-related adverse events (71% vs. 45%), most commonly resulting from myelosuppression. These were more frequent and severe in patients with hematologic malignancies; 22 patients exhibited tumor lysis syndrome. Overall response rates at the RDEs were 10.3% [95% confidence interval (CI), 2.2-27.4] in solid tumors and 4.2% (95% CI, 0.1-21.1), 20% (95% CI, 4.3-48.1), and 22.2% (95% CI, 8.6-42.3) in acute myeloid leukemia (AML) in 1B, 1A, and 2C, respectively. CONCLUSIONS: A common safety profile was identified and preliminary activity was noted, particularly in AML. Comprehensive investigation of dosing regimens yielded recommended doses/regimens for future combination studies.

Repurposing of the ALK Inhibitor Crizotinib for Acute Leukemia and Multiple Myeloma Cells.

Crizotinib was a first generation of ALK tyrosine kinase inhibitor approved for the treatment of ALK-positive non-small-cell lung carcinoma (NSCLC) patients. COMPARE and cluster analyses of transcriptomic data of the NCI cell line panel indicated that genes with different cellular functions regulated the sensitivity or resistance of cancer cells to crizotinib. Transcription factor binding motif analyses in gene promoters divulged two transcription factors possibly regulating the expression of these genes, i.e., RXRA and GATA1, which are important for leukemia and erythroid development, respectively. COMPARE analyses also implied that cell lines of various cancer types displayed varying degrees of sensitivity to crizotinib. Unexpectedly, leukemia but not lung cancer cells were the most sensitive cells among the different types of NCI cancer cell lines. Re-examining this result in another panel of cell lines indeed revealed that crizotinib exhibited potent cytotoxicity towards acute myeloid leukemia and multiple myeloma cells. P-glycoprotein-overexpressing CEM/ADR5000 leukemia cells were cross-resistant to crizotinib. NCI-H929 multiple myeloma cells were the most sensitive cells. Hence, we evaluated the mode of action of crizotinib on these cells. Although crizotinib is a TKI, it showed highest correlation rates with DNA topoisomerase II inhibitors and tubulin inhibitors. The altered gene expression profiles after crizotinib treatment predicted several networks, where TOP2A and genes related to cell cycle were downregulated. Cell cycle analyses showed that cells incubated with crizotinib for 24 h accumulated in the G2M phase. Crizotinib also increased the number of p-H3(Ser10)-positive NCI-H929 cells illustrating crizotinib's ability to prevent mitotic exit. However, cells accumulated in the sub-G0G1 fraction with longer incubation periods, indicating apoptosis induction. Additionally, crizotinib disassembled the tubulin network of U2OS cells expressing an α-tubulin-GFP fusion protein, preventing migration of cancer cells. This result was verified by in vitro tubulin polymerization assays. In silico molecular docking also revealed a strong binding affinity of crizotinib to the colchicine and Vinca alkaloid binding sites. Taken together, these results demonstrate that crizotinib destabilized microtubules. Additionally, the decatenation assay showed that crizotinib partwise inhibited the catalytic activity of DNA topoisomerase II. In conclusion, crizotinib exerted kinase-independent cytotoxic effects through the dual inhibition of tubulin polymerization and topoisomerase II and might be used to treat not only NSCLC but also multiple myeloma.

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.

Homozygous BCMA gene deletion in response to anti-BCMA CAR T cells in a patient with multiple myeloma.

B cell maturation antigen (BCMA) is a target for various immunotherapies and a biomarker for tumor load in multiple myeloma (MM). We report a case of irreversible BCMA loss in a patient with MM who was enrolled in the KarMMa trial ( NCT03361748 ) and progressed after anti-BCMA CAR T cell therapy. We identified selection of a clone with homozygous deletion of TNFRSF17 (BCMA) as the underlying mechanism of immune escape. Furthermore, we found heterozygous TNFRSF17 loss or monosomy 16 in 37 out of 168 patients with MM, including 28 out of 33 patients with hyperhaploid MM who had not been previously treated with BCMA-targeting therapies, suggesting that heterozygous TNFRSF17 deletion at baseline could theoretically be a risk factor for BCMA loss after immunotherapy.

Pasotuxizumab, a BiTE® immune therapy for castration-resistant prostate cancer: Phase I, dose-escalation study findings.

Aim: We report results of a first-in-human study of pasotuxizumab, a PSMA bispecific T-cell engager (BiTE®) immune therapy mediating T-cell killing of tumor cells in patients with advanced castration-resistant prostate cancer. Patients & methods: We assessed once-daily subcutaneous (SC) pasotuxizumab. All SC patients developed antidrug antibodies; therefore, continuous intravenous (cIV) infusion was assessed. Results: A total of 47 patients received pasotuxizumab (SC: n = 31, 0.5-172 µg/d; cIV: n = 16, 5-80 µg/d). The SC maximum tolerated dose was 172.0 µg/d. A sponsor change stopped the cIV cohort early; maximum tolerated dose was not determined. PSA responders occurred (>50% PSA decline: SC, n = 9; cIV, n = 3), including two long-term responders. Conclusion: Data support pasotuxizumab safety in advanced castration-resistant prostate cancer and represent evidence of BiTE monotherapy efficacy in solid tumors. Clinical trial registration: NCT01723475 (ClinicalTrials.gov).

Targeting of the E3 ubiquitin-protein ligase HUWE1 impairs DNA repair capacity and tumor growth in preclinical multiple myeloma models.

Experimental evidence suggests that ubiquitin-protein ligases regulate a number of cellular processes involved in tumorigenesis. We analysed the role of the E3 ubiquitin-protein ligase HUWE1 for pathobiology of multiple myeloma (MM), a still incurable blood cancer. mRNA expression analysis indicates an increase in HUWE1 expression levels correlated with advanced stages of myeloma. Pharmacologic as well as RNAi-mediated HUWE1 inhibition caused anti-proliferative effects in MM cell lines in vitro and in an MM1.S xenotransplantation mouse model. Cell cycle analysis upon HUWE1 inhibition revealed decreased S phase cell fractions. Analyses of potential HUWE1-dependent molecular functions did not show involvement in MYC-dependent gene regulation. However, HUWE1 depleted MM cells displayed increased DNA tail length by comet assay, as well as changes in the levels of DNA damage response mediators such as pBRCA1, DNA-polymerase ß, γH2AX and Mcl-1. Our finding that HUWE1 might thus be involved in endogenous DNA repair is further supported by strongly enhanced apoptotic effects of the DNA-damaging agent melphalan in HUWE1 depleted cells in vitro and in vivo. These data suggest that HUWE1 might contribute to tumour growth by endogenous repair of DNA, and could therefore potentially be exploitable in future treatment developments.

RAL GTPases mediate multiple myeloma cell survival and are activated independently of oncogenic RAS.

Oncogenic RAS provides crucial survival signaling for up to half of multiple myeloma cases, but has so far remained a clinically undruggable target. RAL is a member of the RAS superfamily of small GTPases and is considered to be a potential mediator of oncogenic RAS signaling. In primary multiple myeloma, we found RAL to be overexpressed in the vast majority of samples when compared with pre-malignant monoclonal gammopathy of undetermined significance or normal plasma cells. We analyzed the functional effects of RAL abrogation in myeloma cell lines and found that RAL is a critical mediator of survival. RNAi-mediated knockdown of RAL resulted in rapid induction of tumor cell death, an effect which was independent from signaling via mitogen-activated protein kinase, but appears to be partially dependent on Akt activity. Notably, RAL activation was not correlated with the presence of activating RAS mutations and remained unaffected by knockdown of oncogenic RAS. Furthermore, transcriptome analysis yielded distinct RNA expression signatures after knockdown of either RAS or RAL. Combining RAL depletion with clinically relevant anti-myeloma agents led to enhanced rates of cell death. Our data demonstrate that RAL promotes multiple myeloma cell survival independently of oncogenic RAS and, thus, this pathway represents a potential therapeutic target in its own right.

MOR202, a novel anti-CD38 monoclonal antibody, in patients with relapsed or refractory multiple myeloma: a first-in-human, multicentre, phase 1-2a trial.

BACKGROUND: Treatment of multiple myeloma is not curative, but targeting CD38 improves patient survival. To further explore this therapeutic approach, we investigated the safety and activity of MOR202, a novel monoclonal antibody targeting CD38, in patients with multiple myeloma. METHODS: This is a multicentre, open-label, phase 1-2a trial done at ten hospitals in Germany and Austria. Enrolled patients were aged 18 years or older with relapsed or refractory multiple myeloma and Karnofsky performance status of 60% or higher. Patients were assigned to the different treatment regimens with MOR202 ranging between 0·01 mg/kg and 16 mg/kg in a 3 + 3 design. Dose-escalation and expansion was done either with MOR202 intravenous infusions alone (MOR202 q2w [twice a week] and q1w [weekly] groups) or in combination with dexamethasone (MOR202 with dexamethasone group), with dexamethasone plus pomalidomide (MOR202 with dexamethasone plus pomalidomide group) or plus lenalidomide (MOR202 with dexamethasone plus lenalidomide group). Primary endpoints were safety, MOR202 maximum tolerated dose (or recommended dose) and regimen, and immunogenicity. The primary analysis was assessed in the safety population, which included patients who received at least one dose of any study drug. This trial is registered with ClinicalTrials.gov, NCT01421186. FINDINGS: Between Aug 24, 2011, and Aug 1, 2017, 91 patients were treated, 35 with MOR202 monotherapy, and 56 with MOR202 combination regimens (18 in the MOR202 with dexamethasone group, 21 in the MOR202 with dexamethasone plus pomalidomide group, and 17 in the MOR202 with dexamethasone plus lenalidomide group). MOR202 intravenous infusions were safely administered within 30 min. Infusion-related reactions occurred in 14 (40%) of 35 patients receiving MOR202 monotherapy without steroids, and in four (7%) of 56 patients receiving MOR202 combination treatment. MOR202 maximum tolerated dose was not reached and the recommended regimens were MOR202 administered as an intravenous infusion for 30 min at doses up to 16 mg/kg with dexamethasone (40 mg), or in combination with dexamethasone plus lenalidomide (25 mg) or pomalidomide (4 mg). 35 (38%) of 91 patients developed lymphopenia, 30 (33%) developed neutropenia, and 27 (30%) developed leukopenia; these were the most common grade 3 or higher treatment-emergent adverse events. Serious adverse events were reported in 51 (56%) of 91 patients. None of the deaths were associated with MOR202. One pomalidomide-associated death occurred in the MOR202 with dexamethasone plus pomalidomide group. No anti-MOR202 antibodies were detected in patients. INTERPRETATION: MOR202 is safe and its clinical activity in patients with relapsed or refractory multiple myeloma is promising. Further clinical investigations of combinations with an immunomodulatory drug and dexamethasone are recommended. FUNDING: MorphoSys AG.

The heat shock protein 70 inhibitor VER155008 suppresses the expression of HSP27, HOP and HSP90ß and the androgen receptor, induces apoptosis, and attenuates prostate cancer cell growth.

Heat shock proteins (HSPs) are molecular chaperones that play a pivotal role in correct folding, stabilization and intracellular transport of many client proteins including those involved in oncogenesis. HSP70, which is frequently overexpressed in prostate cancer (PCa), has been shown to critically contribute to tumor cell survival, and might therefore represent a potential therapeutic target. We treated both the androgen receptor (AR)-positive LNCaP and the AR-negative PC-3 cell lines with the pharmacologic HSP70 inhibitor VER155008. Although we observed antiproliferative effects and induction of apoptosis upon HSP70 inhibition, the apoptotic effect was more pronounced in AR-positive LNCaP cells. In addition, VER155008 treatment induced G1 cell cycle arrest in LNCaP cells and decreased AR expression. Further analysis of the HSP system by Western blot analysis revealed that expression of HSP27, HOP and HSP90ß was significantly inhibited by VER155008 treatment, whereas the HSP40, HSP60, and HSP90α expression remained unchanged. Taken together, VER155008 might serve as a novel therapeutic option in PCa patients independent of the AR expression status.

Long-term outcome of patients with relapsed/refractory B-cell non-Hodgkin lymphoma treated with blinatumomab.

Blinatumomab, the first-in-class CD3/CD19 bispecific T-cell engager antibody construct, has recently been approved for treating patients with relapsed or refractory (R/R) B-cell acute lymphoblastic leukemia. However, the clinical proof of concept of blinatumomab efficacy was initially demonstrated in patients with R/R B-cell non-Hodgkin lymphoma (B-NHL) in the MT103-104 phase 1 dose-escalation and expansion trial (NCT00274742), which defined 60 µg/m2 per day as the maximum tolerated dose (MTD). The clinically most relevant adverse effects were neurologic symptoms and cytokine release syndrome. Currently, there are no data on long-term outcomes and toxicity for B-NHL patients receiving blinatumomab treatment, so we performed a single-center, long-term follow-up analysis of 38 patients who participated in the MT103-104 phase 1 trial. We found no evidence for long-term toxicities, especially no blinatumomab-induced neurocognitive impairments. For the entire study population, the median overall survival (OS) was 4.6 years. Remarkably, patients who had received ≥60 µg/m2 per day and responded to blinatumomab achieved a median OS of 7.7 years. Of note, 6 of the surviving patients treated at the MTD have been treatment-free for more than 7 years. In contrast, patients who were treated at dose levels below the MTD had a median OS of only 1.1 years. These results indicate that 60 µg/m2 per day seems to represent the targeted dose level of blinatumomab required for durable remission in R/R B-NHL. Here, we provide the first clinical evidence that blinatumomab lacks long-term toxicity and has the potential to induce sustained remissions in patients with R/R B-NHL.

Novel cell line models to study mechanisms and overcoming strategies of proteasome inhibitor resistance in multiple myeloma.

Experimental data on resistance mechanisms of multiple myeloma (MM) to ixazomib (IXA), a second-generation proteasome inhibitor (PI), are currently lacking. We generated MM cell lines with a 10-fold higher resistance to IXA as their sensitive counterparts, and observed cross-resistance towards the PIs carfilzomib (CFZ) and bortezomib (BTZ). Analyses of the IXA-binding proteasome subunits PSMB5 and PSMB1 show increased PSMB5 expression and activity in all IXA-resistant MM cells, and upregulated PSMB1 expression in IXA-resistant AMO1 cells. In addition, sequence analysis of PSMB5 revealed a p.Thr21Ala mutation in IXA-resistant MM1.S cells, and a p.Ala50Val mutation in IXA-resistant L363 cells, whereas IXA-resistant AMO1 cells lack PSMB5 mutations. IXA-resistant cells retain their sensitivity to therapeutic agents that mediate cytotoxic effects via induction of proteotoxic stress. Induction of ER stress and apoptosis by the p97 inhibitor CB-5083 was strongly enhanced in combination with the PI3Kα inhibitor BYL-719 or the HDAC inhibitor panobinostat suggesting potential therapeutic strategies to circumvent IXA resistance in MM. Taken together, our newly established IXA-resistant cell lines provide first insights into resistance mechanisms and overcoming treatment strategies, and represent suitable models to further study IXA resistance in MM.

Spectrum and functional validation of PSMB5 mutations in multiple myeloma.

Despite an increasing number of approved therapies, multiple myeloma (MM) remains an incurable disease and only a small number of patients achieve prolonged disease control. Some genes have been linked with response to commonly used anti-MM compounds, including immunomodulators (IMiDs) and proteasome inhibitors (PIs). In this manuscript, we demonstrate an increased incidence of acquired proteasomal subunit mutations in relapsed MM compared to newly diagnosed disease, underpinning a potential role of point mutations in the clonal evolution of MM. Furthermore, we are first to present and functionally characterize four somatic PSMB5 mutations from primary MM cells identified in a patient under prolonged proteasome inhibition, with three of them affecting the PI-binding pocket S1. We confirm resistance induction through missense mutations not only to Bortezomib, but also, in variable extent, to the next-generation PIs Carfilzomib and Ixazomib. In addition, a negative impact on the proteasome activity is assessed, providing a potential explanation for later therapy-induced eradication of the affected tumor subclones in this patient.