Proteasome Inhibitor Bortezomib Research Articles

Enhanced angiogenesis of human umbilical vein endothelial cells via THP-1-derived M2c-like macrophages and treatment with proteasome inhibitors 'bortezomib and ixazomib'.

The leading cause of cancer-related death is lung cancer, with metastasis being the most common cause of death. To elucidate the role of macrophages in lung cancer and angiogenesis processes, we established an in vitro co-culture model of A549 or HUVEC with THP-1 cells that polarized to M2c macrophages with hydrocortisone. The proteasome inhibitors bortezomib and ixazomib were investigated for their effects on proliferation, invasion, migration, metastasis, and angiogenesis pathways. The effects of bortezomib and ixazomib on gene expression in gene panels, including crucial genes related to angiogenesis and proteasomes, were investigated after the co-culture model to determine these effects at the molecular level. In conclusion, bortezomib and ixazomib showed antiproliferative effects in both cells, as well as in M2c macrophage co-culture. M2c macrophages also increased invasion in A549 cells and both invasion and migration in HUVEC. mRNA expression upregulation, specifically in the NFKB and VEGF genes, supported the metastatic and angiogenic effects found in A549 and HUVEC with M2c macrophage co-culture. Additionally, bortezomib inhibited the VEGFB pathway in HUVEC and NFKB1 in A549 cells. The significant findings obtained as a result of this study will provide information regarding angiogenesis induced by M2 macrophages.

Reduction of Filamin C Results in Altered Proteostasis, Cardiomyopathy, and Arrhythmias.

Many cardiomyopathy-associated FLNC pathogenic variants are heterozygous truncations, and FLNC pathogenic variants are associated with arrhythmias. Arrhythmia triggers in filaminopathy are incompletely understood. We describe an individual with biallelic FLNC pathogenic variants, p.Arg650X and c.970-4A>G, with peripartum cardiomyopathy and ventricular arrhythmias. We also describe clinical findings in probands with FLNC variants including Val2715fs87X, Glu2458Serfs71X, Phe106Leu, and c.970-4A>G with hypertrophic and dilated cardiomyopathy, atrial fibrillation, and ventricular tachycardia. Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) were generated. The FLNC truncation, Arg650X/c.970-4A>G, showed a marked reduction in filamin C protein consistent with biallelic loss of function mutations. To assess loss of filamin C, gene editing of a healthy control iPSC line was used to generate a homozygous FLNC disruption in the actin binding domain. Because filamin C has been linked to protein quality control, we assessed the necessity of filamin C in iPSC-CMs for response to the proteasome inhibitor bortezomib. After exposure to low-dose bortezomib, FLNC-null iPSC-CMs showed an increase in the chaperone proteins BAG3, HSP70 (heat shock protein 70), and HSPB8 (small heat shock protein B8) and in the autophagy marker LC3I/II. FLNC null iPSC-CMs had prolonged electric field potential, which was further prolonged in the presence of low-dose bortezomib. FLNC null engineered heart tissues had impaired function after low-dose bortezomib. FLNC pathogenic variants associate with a predisposition to arrhythmias, which can be modeled in iPSC-CMs. Reduction of filamin C prolonged field potential, a surrogate for action potential, and with bortezomib-induced proteasome inhibition, reduced filamin C led to greater arrhythmia potential and impaired function.

Interleukin-33 increases the sensitivity of multiple myeloma cells to the proteasome inhibitor bortezomib through reactive oxygen species-mediated inhibition of nuclear factor kappa-B signal and stemness properties.

The proteasome inhibitor bortezomib (BTZ) is the first-line therapy for multiple myeloma (MM). BTZ resistance largely limits its clinical application in MM. Interleukin-33 (IL-33) exerts antitumor effects through various mechanisms, including enhancing antitumor immunity and promoting the apoptosis of cancer cells. Here, the synergistic anti-MM effect of IL-33 and BTZ was verified, and the underlying mechanisms were elucidated. Bioinformatic analysis indicated that IL-33 expression levels were downregulated in MM, and that BTZ-treated MM patients with high IL-33 levels had better prognosis than those with low IL-33 levels. Moreover, the patients with high IL-33 levels had a better treatment response to BTZ. Further immune analysis suggested that IL-33 can enhance the anti-MM immunity. IL-33 and BTZ synergistically inhibited proliferation and induced apoptosis of MM cells, which was mediated by the excessive accumulation of cellular reactive oxygen species (ROS). Furthermore, increased ROS hindered the nuclear translocation of NF-κB-p65, thereby decreasing the transcription of target stemness-related genes (SOX2, MYC, and OCT3/4). These effects induced by the combination therapy could be reversed by eliminating ROS by N-acetylcysteine. In conclusion, our results indicated that IL-33 enhanced the sensitivity of MM to BTZ through ROS-mediated inhibition of nuclear factor kappa-B (NF-κB) signal and stemness properties.

Proteasome inhibitor bortezomib prevents proliferation and migration of pulmonary arterial smooth muscle cells.

Pulmonary vascular remodeling is a key pathological process of pulmonary arterial hypertension (PAH), characterized by uncontrolled proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs). Bortezomib (BTZ) is the first Food and Drug Administration (FDA)-approved proteasome inhibitor for multiple myeloma treatment. Recently, there is emerging evidence showing its effect on reversing PAH, although its mechanisms are not well understood. In this study, anti-proliferative and anti-migratory effects of BTZ on PASMCs were first examined by different inducers such as fetal bovine serum (FBS), angiotensin II (Ang II) and platelet-derived growth factor (PDGF)-BB, while potential mechanisms including cellular reactive oxygen species (ROS) and mitochondrial ROS were then investigated; finally, signal transduction of ERK and Akt was examined. Our results showed that BTZ attenuated FBS-, Ang II- and PDGF-BB-induced proliferation and migration, with associated decreased cellular ROS production and mitochondrial ROS production. In addition, the phosphorylation of ERK and Akt induced by Ang II and PDGF-BB was also inhibited by BTZ treatment. This study indicates that BTZ can prevent proliferation and migration of PASMCs, which are possibly mediated by decreased ROS production and down-regulation of ERK and Akt. Thus, proteasome inhibition can be a novel pharmacological target in the management of PAH.

Abstract P05: Novel PROTAC-based EZH2 Degraders Effectively Target Lymphoid Malignancies by Depleting Enzyme-Independent Functions of EZH2

Abstract Overexpression or mutation of the epigenetic silencer EZH2 correlates with invasive cancer growth, resistance, and poor survival outcomes. Previously, we have established a methylation-independent role for EZH2 in the transcriptional activation of oncogenes in NKTL. As EZH2 enzymatic inhibitors have proven ineffective against NKTL, we embarked on the development of novel EZH2 Proteolysis Targeting Chimera (PROTAC) compounds. EZH2 PROTACs consist of an EZH2- and E3-ligase binding domain which promotes targeted ubiquitination of the EZH2 protein thereby marking it for proteasomal degradation. Our initial efforts focused on the iterative synthesis of EZH2 PROTAC candidates optimizing factors such as permeability, solubility and stable ternary complex formation. Through extensive screening, we identified C2911 and C1311, which successfully trigger the dose-dependent degradation of EZH2 and PRC2 components concomitant with a significant loss of survival and induction of apoptosis in both NKTL and DLBCL. These effects were rescued by pre-treatment with proteasome inhibitor Bortezomib or when E3 ligase activity was disrupted through siRNA knockdown. In NKTL, we show that EZH2 PROTAC treatment decreased JAK3 and phospho-STAT5 protein expression and identified the suppression of the JAK-STAT-MYC axis as a potential mechanism of action. EZH2 PROTAC treatment effectively reactivates transcription of canonical EZH2 target genes such as MYT1 and ANPEP and downregulates expression of oncogenes c-Myc and E2F1. Pharmacokinetic studies in vivo demonstrate that these PROTACs exhibit good plasma stability, half-life (>4h) and low clearance thereby establishing potential for future clinical development. The efficacy of EZH2 PROTACs in xenograft mice are currently being evaluated. Collectively, our data supports the discovery of novel EZH2 PROTACs and the critical role it plays in successfully targeting the oncogenic role of EZH2 in NKTL or other non-canonical EZH2-driven cancers through protein depletion where enzymatic-only inhibition was ineffective. We are further investigating the mechanisms stabilizing EZH2 protein levels to enhance PROTAC-mediated cytotoxicity. Citation Format: Nurulhuda Mustafa, Zubaida Talal Alnaseri, Darrel Tan Egk Jian, David Quach Thanh Truong, Wan Hsin Lim, Wee Joo Chng. Novel PROTAC-based EZH2 Degraders Effectively Target Lymphoid Malignancies by Depleting Enzyme-Independent Functions of EZH2 [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr P05.

Chromatin Profiles Are Prognostic of Clinical Response to Bortezomib-Containing Chemotherapy in Pediatric Acute Myeloid Leukemia: Results from the COG AAML1031 Trial

The addition of the proteasome inhibitor bortezomib to standard chemotherapy did not improve survival in pediatric acute myeloid leukemia (AML) when all patients were analyzed as a group in the Children’s Oncology Group phase 3 trial AAML1031 (NCT01371981). Proteasome inhibition influences the chromatin landscape and proteostasis, and we hypothesized that baseline proteomic analysis of histone- and chromatin-modifying enzymes (HMEs) would identify AML subgroups that benefitted from bortezomib addition. A proteomic profile of 483 patients treated with AAML1031 chemotherapy was generated using a reverse-phase protein array. A relatively high expression of 16 HME was associated with lower EFS and higher 3-year relapse risk after AML standard treatment compared to low expressions (52% vs. 29%, p = 0.005). The high-HME profile correlated with more transposase-accessible chromatin, as demonstrated via ATAC-sequencing, and the bortezomib addition improved the 3-year overall survival compared with standard therapy (62% vs. 75%, p = 0.033). These data suggest that there are pediatric AML populations that respond well to bortezomib-containing chemotherapy.

Synergistic Effect of a Combination of Proteasome and Ribonucleotide Reductase Inhibitors in a Biochemical Model of the Yeast Saccharomyces cerevisiae and a Glioblastoma Cell Line.

Proteasome inhibitors are used in the therapy of several cancers, and clinical trials are underway for their use in the treatment of glioblastoma (GBM). However, GBM becomes resistant to chemotherapy relatively rapidly. Recently, the overexpression of ribonucleotide reductase (RNR) genes was found to mediate therapy resistance in GBM. The use of combinations of chemotherapeutic agents is considered a promising direction in cancer therapy. The present work aimed to evaluate the efficacy of the combination of proteasome and RNR inhibitors in yeast and GBM cell models. We have shown that impaired proteasome function results in increased levels of RNR subunits and increased enzyme activity in yeast. Co-administration of the proteasome inhibitor bortezomib and the RNR inhibitor hydroxyurea was found to significantly reduce the growth rate of S. cerevisiae yeast. Accordingly, the combination of bortezomib and another RNR inhibitor gemcitabine reduced the survival of DBTRG-05MG compared to the HEK293 cell line. Thus, yeast can be used as a simple model to evaluate the efficacy of combinations of proteasome and RNR inhibitors.

Loss of GABARAP mediates resistance to immunogenic chemotherapy in multiple myeloma

AbstractImmunogenic cell death (ICD) is a form of cell death by which cancer treatments can induce a clinically relevant antitumor immune response in a broad range of cancers. In multiple myeloma (MM), the proteasome inhibitor bortezomib is an ICD inducer and creates durable therapeutic responses in patients. However, eventual relapse and resistance to bortezomib appear inevitable. Here, by integrating patient transcriptomic data with an analysis of calreticulin (CRT) protein interactors, we found that GABA type A receptor–associated protein (GABARAP) is a key player whose loss prevented tumor cell death from being perceived as immunogenic after bortezomib treatment. GABARAP is located on chromosome 17p, which is commonly deleted in patients with high risk MM. GABARAP deletion impaired the exposure of the eat-me signal CRT on the surface of dying MM cells in vitro and in vivo, thus reducing tumor cell phagocytosis by dendritic cells and the subsequent antitumor T-cell response. Low GABARAP was independently associated with shorter survival in patients with MM and reduced tumor immune infiltration. Mechanistically, we found that GABARAP deletion blocked ICD signaling by decreasing autophagy and altering Golgi apparatus morphology, with consequent defects in the downstream vesicular transport of CRT. Conversely, upregulating autophagy using rapamycin restored Golgi morphology, CRT exposure, and ICD signaling in GABARAPKO cells undergoing bortezomib treatment. Therefore, coupling an ICD inducer, such as bortezomib, with an autophagy inducer, such as rapamycin, may improve patient outcomes in MM, in which low GABARAP in the form of del(17p) is common and leads to worse outcomes.

Abstract 7191: Targeting IGF1R/INSR pathway with approved ALK inhibitors inhibits AKT signaling and overcomes proteasome inhibitor resistance in multiple myeloma

Abstract Background: Resistance to proteasome inhibitors (PI) bortezomib (BTZ) and carfilzomib (CFZ) is a major obstacle to the successful treatment of multiple myeloma (MM); thus, identification of novel therapeutic options is an unmet medical need. ALK inhibitors have been shown to exhibit anti-MM activity in monotherapy or in combination with CFZ. Our data showed that MM cells were negative for ALK; thus, we aimed to identify the mechanism of action of the ALK inhibitor ceritinib in MM cells and the mechanism of synergy between ceritinib and CFZ. Methods: A set of PI-naïve and PI-resistant MM cells was used in this study. Genome-wide CRISPR/Cas9-based screening using the Brunello library was performed in the AMO-1 cell line. Kinase inhibitor selectivity data were retrieved from ChEMBL, v30 database. RNA sequencing was used to determine expression changes after treatment, and RNA-seq data from patients included in the CoMMpass study were analyzed. Western blotting was performed to assess the levels of total and phosphorylated protein. Unbiased LC-MS/MS was performed to determine the effects of ceritinib and CFZ on the intracellular metabolites. An in vivo mouse model based on the orthotopic injection of AMO-BTZ cells into the femur of NSG mice was used to determine the effect of the drug combination in vivo. Results: Among approved ALK inhibitors, ceritinib has been identified as the most synergistic cytotoxic combination with CFZ in PI-naïve and PI-resistant MM cells. An in silico search of the ChEMBL database identified InsR and IGF1R as receptor tyrosine kinase that are inhibited by ceritinib. PI-naïve, PI-resistant cell lines, as well as MM patients were negative for ALK, but positive for InsR and IGF1R expression. CRISPR/Cas9-screening identified genes involved in the negative regulation of mTORC signaling (DDIT4, NPRL2/3, TSC1/2) and the transcription factor FOXO1 as the major resistance candidates to ceritinib. Subsequently, FOXO1 inhibition using a selective chemical inhibitor protected the cells from ceritinib-induced cytotoxicity. Next, ceritinib inhibited mTORC signaling and induced the expression of genes related to cell cycle arrest, which are downstream of FOXO1. Likewise, ceritinib, by targeting the InsR/IGF1R, impaired purine-pyrimidine and amino acid homeostasis, suggesting metabolic and proliferation shut-down and amino acid starvation due to downstream mTORC and Akt inhibition and FOXO1 induction. The combination of ceritinib and CFZ was superior to CFZ in PI-resistant MM in vivo and showed strong synergistic cytotoxicity in primary cells from MM patients progressing under or after PI-containing therapy. Conclusion: Ceritinib, an FDA-approved drug, overcomes PI resistance in MM by inhibiting InsR/IGF1R and downstream Akt signaling. Therefore, ceritinib is a promising treatment option for PI-resistant MM. Supported by the project National Institute for Cancer Research (Programme EXCELES, ID Project No. LX22NPO5102) - Funded by the European Union - Next Generation EU. Citation Format: Andrej Besse, Tiberiu Totu, Marianne Kraus, Anthonius P. Janssen, Jana Veprkova, Max Mendez Lopez, Ondrej Slaby, Marija Buljan, Mario van der Stelt, Christoph Driessen, Lenka Besse. Targeting IGF1R/INSR pathway with approved ALK inhibitors inhibits AKT signaling and overcomes proteasome inhibitor resistance in multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7191.

Expression of the chemokine receptor CCR1 decreases sensitivity to bortezomib in multiple myeloma cell lines

BackgroundThe proteasome inhibitor bortezomib is one of the primary therapies used for the haematological malignancy multiple myeloma (MM). However, intrinsic or acquired resistance to bortezomib, via mechanisms that are not fully elucidated, is a barrier to successful treatment in many patients. Our previous studies have shown that elevated expression of the chemokine receptor CCR1 in MM plasma cells in newly diagnosed MM patients is associated with poor prognosis. Here, we hypothesised that the poor prognosis conferred by CCR1 expression is, in part, due to a CCR1-mediated decrease in MM plasma cell sensitivity to bortezomib. MethodsIn order to investigate the role of CCR1 in MM cells, CCR1 was knocked out in human myeloma cell lines OPM2 and U266 using CRISPR-Cas9. Additionally, CCR1 was overexpressed in the mouse MM cell line 5TGM1. The effect of bortezomib on CCR1 knockout or CCR1-overexpressing cells was then assessed by WST-1 assay, with or without CCL3 siRNA knockdown or addition of recombinant human CCL3. NSG mice were inoculated intratibially with OPM2-CCR1KO cells and were treated with 0.7 mg/kg bortezomib or vehicle twice per week for 3 weeks and GFP+ tumour cells in the bone marrow were quantitated by flow cytometry. The effect of CCR1 overexpression or knockout on unfolded protein response pathways was assessed using qPCR for ATF4, HSPA5, XBP1, ERN1 and CHOP and Western blot for IRE1α and p-Jnk. ResultsUsing CCR1 overexpression or CRIPSR-Cas9-mediated CCR1 knockout in MM cell lines, we found that CCR1 expression significantly decreases sensitivity to bortezomib in vitro, independent of the CCR1 ligand CCL3. In addition, CCR1 knockout rendered the human MM cell line OPM2 more sensitive to bortezomib in an intratibial MM model in NSG mice in vivo. Moreover, CCR1 expression negatively regulated the expression of the unfolded protein response receptor IRE1 and downstream target gene XBP1, suggesting this pathway may be responsible for the decreased bortezomib sensitivity of CCR1-expressing cells. ConclusionsTaken together, these studies suggest that CCR1 expression may be associated with decreased response to bortezomib in MM cell lines.

Abstract P24: GABARAP deficiency drives resistance to immunogenic chemotherapy in multiple myeloma

Abstract P24: GABARAP deficiency drives resistance to immunogenic chemotherapy in multiple myeloma

Interferon and B-cell Signatures Inform Precision Medicine in Lupus Nephritis

IntroductionCurrent therapeutic management of lupus nephritis (LN) fails to induce long-term remission in over 50% of patients, highlighting the urgent need for additional options. MethodsWe analysed differentially expressed genes in peripheral blood from active LN (n=41) and active non-renal lupus (n=62) patients versus healthy controls (n=497) from the European PRECISESADS project (NTC02890121), and dysregulated gene modules in a discovery (n=26) and a replication (n=15) set of active LN cases. ResultsReplicated gene modules qualified for correlation analyses with serological markers, and regulatory network and druggability analysis. Unsupervised co-expression network analysis revealed 20 dysregulated gene modules and stratified the active LN population into three distinct subgroups. These subgroups were characterised by low, intermediate, and high interferon (IFN) signatures, with differential dysregulation of the “B cell” and “plasma cells/immunoglobulins” modules. Drugs annotated to the IFN network included CC-motif chemokine receptor 1 inhibitors, programmed death-ligand 1 inhibitors, and irinotecan, while the anti-CD38 daratumumab and proteasome inhibitor bortezomib showed potential for counteracting the “plasma cells/immunoglobulins” transcriptomic signature. In silico analysis demonstrated the low-IFN subgroup to benefit from calcineurin inhibition and the intermediate-IFN subgroup from B cell targeted therapies. High-IFN patients exhibited greater anticipated response to anifrolumab while daratumumab appeared beneficial for the intermediate-IFN and high-IFN subgroups. ConclusionIn summary, IFN upregulation and B and plasma cell gene dysregulation patterns revealed three LN patient subgroups, which may not necessarily represent distinct disease phenotypes but rather phases of the inflammatory processes during a renal flare, providing a conceptual framework for precision medicine in LN.

Preclinical efficacy of carfilzomib in BRAF-mutant colorectal cancer models.

Serine/threonine-protein kinase B-raf (BRAF) mutations are found in 8-15% of colorectal cancer patients and identify a subset of tumors with poor outcome in the metastatic setting. We have previously reported that BRAF-mutant human cells display a high rate of protein production, causing proteotoxic stress, and are selectively sensitive to the proteasome inhibitors bortezomib and carfilzomib. In this work, we tested whether carfilzomib could restrain the growth of BRAF-mutant colorectal tumors not only by targeting cancer cells directly, but also by promoting an immune-mediated antitumor response. In human and mouse colorectal cancer cells, carfilzomib triggered robust endoplasmic reticulum stress and autophagy, followed by the emission of immunogenic-damage-associated molecules. Intravenous administration of carfilzomib delayed the growth of BRAF-mutant murine tumors and mobilized the danger-signal proteins calreticulin and high mobility group box 1 (HMGB1). Analyses of drug-treated samples revealed increased intratumor recruitment of activated cytotoxic T cells and natural killers, concomitant with the downregulation of forkhead box protein P3 (Foxp3)+ T-cell surface glycoprotein CD4 (CD4)+ T cells, indicating that carfilzomib promotes reshaping of the immune microenvironment of BRAF-mutant murine colorectal tumors. These results will inform the design of clinical trials in BRAF-mutant colorectal cancer patients.

Imaging Very Late Antigen-4 on MOLT4 Leukemia Tumors with Cysteine Site-Specific 89Zr-Labeled Natalizumab Immuno-Positron Emission Tomography.

Very late antigen-4 (VLA4; CD49d) is a promising immune therapy target in treatment-resistant leukemia and multiple myeloma, and there is growing interest in repurposing the humanized monoclonal antibody (Ab), natalizumab, for this purpose. Positron emission tomography with radiolabeled Abs (immuno-PET) could facilitate this effort by providing information on natalizumab's in vivo pharmacokinetic and target delivery properties. In this study, we labeled natalizumab with 89Zr specifically on sulfhydryl moieties via maleimide-deferoxamine conjugation. High VLA4-expressing MOLT4 human T cell acute lymphoblastic leukemia cells showed specific 89Zr-natalizumab binding that was markedly blocked by excess Ab. In nude mice bearing MOLT4 tumors, 89Zr-natalizumab PET showed high-contrast tumor uptake at 7 days postinjection. Biodistribution studies confirmed that uptake was the highest in MOLT4 tumors (2.22 ± 0.41%ID/g) and the liver (2.33 ± 0.76%ID/g), followed by the spleen (1.51 ± 0.42%ID/g), while blood activity was lower at 1.12 ± 0.21%ID/g. VLA4-specific targeting in vivo was confirmed by a 58.1% suppression of tumor uptake (0.93 ± 0.15%ID/g) when excess Ab was injected 1 h earlier. In cultured MOLT4 cells, short-term 3 day exposure to the proteasome inhibitor bortezomib (BTZ) did not affect the α4 integrin level, but BTZ-resistant cells that survived the treatment showed increased α4 integrin expression. When the effects of BTZ treatment were tested in mice, there was no change of the α4 integrin level or 89Zr-natalizumab uptake in MOLT4 leukemia tumors, which underscores the complexity of tumor VLA4 regulation in vivo. In conclusion, 89Zr-natalizumab PET may be useful for noninvasive monitoring of tumor VLA4 and may assist in a more rational application of Ab-based therapies for hematologic malignancies.

A gain-of-function p53 mutant synergizes with oncogenic NRAS to promote acute myeloid leukemia in mice.

We previously demonstrated that a subset of acute myeloid leukemia (AML) patients with concurrent RAS pathway and TP53 mutations have an extremely poor prognosis and that most of these TP53 mutations are missense mutations. Here, we report that, in contrast to the mixed AML and T cell malignancy that developed in NrasG12D/+ p53-/- (NP-/-) mice, NrasG12D/+ p53R172H/+ (NPmut) mice rapidly developed inflammation-associated AML. Under the inflammatory conditions, NPmut hematopoietic stem and progenitor cells (HSPCs) displayed imbalanced myelopoiesis and lymphopoiesis and mostly normal cell proliferation despite MEK/ERK hyperactivation. RNA-Seq analysis revealed that oncogenic NRAS signaling and mutant p53 synergized to establish an NPmut-AML transcriptome distinct from that of NP-/- cells. The NPmut-AML transcriptome showed GATA2 downregulation and elevated the expression of inflammatory genes, including those linked to NF-κB signaling. NF-κB was also upregulated in human NRAS TP53 AML. Exogenous expression of GATA2 in human NPmut KY821 AML cells downregulated inflammatory gene expression. Mouse and human NPmut AML cells were sensitive to MEK and NF-κB inhibition in vitro. The proteasome inhibitor bortezomib stabilized the NF-κB-inhibitory protein IκBα, reduced inflammatory gene expression, and potentiated the survival benefit of a MEK inhibitor in NPmut mice. Our study demonstrates that a p53 structural mutant synergized with oncogenic NRAS to promote AML through mechanisms distinct from p53 loss.

Case report: pomalidomide as maintenance after salvage autologous stem cell transplantation in a patient with relapsed multiple myeloma

Currently, lenalidomide is the only immunomodulatory drug (IMiD) approved for maintenance therapy in patients with newly diagnosed multiple myeloma who have received high-dose chemotherapy and autologous stem cell transplantation (ASCT). The maintenance with lenalidomide showed an advantage over placebo or observation for both progression-free and overall survival in a series of phase 3 randomized trials. Salvage ASCT can be performed after disease relapse in case of a long-term response after the first transplantation or if this option has not been performed before. Pomalidomide is a third-generation IMiD approved for the treatment of relapsed and refractory multiple myeloma, which is efficient in patients with resistance to lenalidomide and proteasome inhibitors. Structurally, lenalidomide and pomalidomide are similar, and therefore the latter can also be considered as a drug for maintenance, however, there are no relevant phase 3 randomized trials. In this article, we present a clinical case of a 60-year-old patient with newly diagnosed multiple myeloma who progressed after 2 lines of induction therapy, which included lenalidomide and two proteasome inhibitors (bortezomib, ixazomib). The use of Pd combination (pomalidomide, dexamethasone) made it possible to achieve a repeated response and implement of salvage ASCT. The second ASCT was carried out only 12 months later after the first due to the COVID-19 pandemic. Subsequent long-term maintenance therapy with pomalidomide resulted in a complete response and minimal residual disease negativity. The resulting response has persisted at the time of this writing for over 2 years. To discuss the presented clinical case, the data of the French phase 2 IFM 2013-01 study were used, in which patients with failed first-line transplantation in case of relapse received PCd (pomalidomide, cyclophosphamide, dexamethasone) induction, salvage ASCT, and maintenance by Pd until disease progression. Pomalidomide may be an acceptable substitute for lenalidomide in patients with prior intolerance or refractory to this IMiD.

A cell‐autonomous role of DNAJC5 in microglial‐mediated proteostasis in Alzheimer’s disease

AbstractBackgroundDNAJC5 encodes co‐chaperone cysteine‐string protein alpha (CSPα), a synaptic and endolysosomal protein whose mutations cause adult‐onset neuronal ceroid lipofuscinosis (ANCL). Characterized by endolysosomal dysfunction, microgliosis, synaptic loss, and protein aggregation, ANCL shares pathological features with Alzheimer’s disease (AD). The endolysosomal role of CSPα in non‐neuronal cell types has been understudied. Here, we aimed to address this gap and hypothesized that CSPα plays a yet undefined microglial role in AD.MethodAβ levels and plaques in human tissue were assessed through ELISA and immunohistochemistry, respectively. Bulk‐RNAseq data from two independent cohorts of AD and ANCL patients with controls (GSE5281; GSE15222) were analyzed. To assess cell‐autonomous expression, single‐nuclei RNAseq (snRNA‐seq) was mined from the parietal cortex of AD patients with APP and PSEN1 mutations (n = 16) along with sporadic AD (n = 31), non‐AD (n = 9), presymptomatic (n = 3), and control patients (n = 8). An independent cohort (GSE138852) was analyzed for rigor. Using Clue.io, candidate therapeutics projected to reverse the observed transcriptomic profile from DNAJC5 mutants in myeloid cells were identified. A cohort from iPSC‐derived microglia (GSE178317) was cross‐analyzed with the ANCL RNAseq.ResultIn tissue from ANCL patients, soluble or insoluble Aβ levels were nearly nonexistent and synaptic protein levels were reduced, but intraneuronal Aβ accumulation was increased, suggesting altered APP processing. In two independent RNAseq cohorts (GSE5281; GSE15222), DNAJC5 was reduced in AD patients versus controls. SnRNA‐seq from parietal cortex revealed higher DNAJC5 transcript levels in microglia versus neurons. RNAseq obtained from ANCL patients was cross‐referenced with dataset obtained from iPSC‐derived microglia, revealing 35 differentially expressed genes associated with proliferative microglia and 32 genes associated with the interferon/chemokine response. Analysis of ANCL transcriptomics in myeloid cell lines using Clue.io indicate HSP inhibitors (BIIB‐021, geldanamycin), an opioid receptor antagonist (BNTX), and a proteasome inhibitor (bortezomib) suggesting convergence in proteostasis in microglial cells. ANCL‐patient transcriptomics displayed reduced TSPAN14, which facilitates ADAM10 maturation, and elevated APH1B and GSAP, enzymes that promote γ‐secretase activity suggesting a shift towards amyloidogenic pathway.ConclusionDNAJC5 is implicated in AD pathogenesis modulating APP processing and amyloidogenesis. Furthermore, endolysosomal DNAJC5 might play a novel cell‐autonomous role in microglia under altered proteostasis.

Biomimetic platelet nanoparticles encapsulated with proteasome inhibitor bortezomib for multiple myeloma treatment

The discovery of bortezomib (BTZ) has been a major clinical breakthrough for multiple myeloma (MM) treatment. However, its clinical application is restricted to a low tumor-targeting ability, fast clearance, and treatment-related toxicity. Here, we report a targeting strategy of MM by poly(lactic-co-glycolic acid)(PLGA) nanoparticles cloaking with platelet membranes (PMs) encapsulating BTZ (PM/BTZNPs). The drug delivery system could encapsulate sufficient BTZ with suitable nanoparticle characteristics for cellular uptake via an easy fabrication process. Of note, PM coating markedly enhances the selectivity, cellular uptake, and anticancer effects of BTZ in LP-1 cells. PM/BTZNPs further display a targeted drug delivery system to MM, causing a low toxicity effect and exhibiting an obvious survival advantage compared to nontargeted BTZ. Therefore, PM/BTZNPs, as a biomimetic nanotherapeutic formulation, demonstrate a high potential for MM patients.

NK92 Expressing Anti-BCMA CAR and Secreted TRAIL for the Treatment of Multiple Myeloma: Preliminary In Vitro Assessment.

Multiple myeloma (MM) has witnessed improved patient outcomes through advancements in therapeutic approaches. Notably, allogeneic stem cell transplantation, proteasome inhibitors, immunomodulatory drugs, and monoclonal antibodies have contributed to enhanced quality of life. Recently, a promising avenue has emerged with chimeric antigen receptor (CAR) T cells targeting B-cell maturation antigen (BCMA), expressed widely on MM cells. To mitigate risks associated with allogenic T cells, we investigated the potential of BCMA CAR expression in natural killer cells (NKs), known for potent cytotoxicity and minimal side effects. Using the NK-92 cell line, we co-expressed BCMA CAR and soluble tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL) employing the piggyBac transposon system. Engineered NK cells (CAR-NK-92-TRAIL) demonstrated robust cytotoxicity against a panel of MM cell lines and primary patient samples, outperforming unmodified NK-92 cells with a mean difference in viability of 45.1% (±26.1%, depending on the target cell line). Combination therapy was explored with the proteasome inhibitor bortezomib (BZ) and γ-secretase inhibitors (GSIs), leading to a significant synergistic effect in combination with CAR-NK-92-TRAIL cells. This synergy was evident in cytotoxicity assays where a notable decrease in MM cell viability was observed in combinatorial therapy compared to single treatment. In summary, our study demonstrates the therapeutic potential of the CAR-NK-92-TRAIL cells for the treatment of MM. The synergistic impact of combining these engineered NK cells with BZ and GSI supports further development of allogeneic CAR-based products for effective MM therapy.

Targeting IGF1R/Insr Pathway with Approved ALK-Inhibitors Overcomes Proteasome Inhibitor Resistance in Multiple Myeloma

Background: The treatment of multiple myeloma (MM) has advanced rapidly with the discovery of proteasome inhibitors (PIs) bortezomib (BTZ) and carfilzomib (CFZ). PIs disrupt the equilibrium between production and disposal of excess and/or misfolded proteins in MM cells, leading to apoptosis. However, despite this success, essentially all patients with MM eventually develop resistance to PIs. Therefore, identifying strategies to overcome PIs resistance is clinically important. ALK inhibitors exhibit anti-MM activity ex vivo. Previously, the ALK inhibitor ceritinib has been shown to inhibit IGF1R/InsR signaling. Signaling through the IGF1/IGF1R axis contributes to acquired resistance to BTZ, and PI activity is reduced in the presence of IGF1, suggesting that IGF1R inhibitors may enhance the cytotoxic effect of PIs. To date, IGF1R/InsR inhibitors have not been successful as single agents in clinical trials and no formally approved drugs are available. Importantly, the drug repurposing approach offers a promising strategy for drug development, particularly for MM. Therefore, we aimed to identify i) whether ALK inhibitors are cytotoxic in MM by targeting the IGF1R/InsR pathway, and ii) whether they can be combined with PIs to overcome PI resistance in vitro and in vivo. Methods: A set of PI-naïve and PI-resistant cells was used in this study. The cytotoxicity of drugs was determined by CCK8 assay in cell lines and by CellTiter-Glo assay in primary cells. Genome-wide CRISPR/Cas9-based loss-of-function screening using the Brunello library was performed in the AMO-1 cell line to identify the mechanism of action of ceritinib. Kinase inhibitor selectivity data were retrieved from ChEMBL, v30. Western blotting was performed to assess the levels of total and phosphorylated proteins. RNA sequencing was used to determine the number of transcripts in PI-sensitive and PI-resistant cells, and RNA-seq data from patients included in the CoMMpass study were analyzed. Unbiased LC-MS/MS was performed to determine the effects of ceritinib and carfilzomib on intracellular metabolites. An in vivo mouse model based on orthotopic injection of AMO-BTZ cells into the femur of NSG mice was used to determine the effect of the drug combination in vivo. Results: Initially, seven ALK inhibitors were tested in PI-naïve and PI-adapted MM cell lines. Based on IC 50 values, the most effective ALK inhibitors to induce cytotoxicity in MM were ceritinib > brigatinib > entrectinib. The combination of ceritinib, brigatinib, and entrectinib showed synergistic cytotoxicity with PI BTZ and CFZ and overcame PI-resistance in four different sets of PI-adapted cells. The strongest synergistic cytotoxicity was observed between ceritinib and CFZ in CFZ-adapted cells. CRISPR/Cas9-screening identified genes involved in the negative regulation of mTORC signaling (DDIT4, NPRL2/3, TSC1/2) and transcription factor FOXO1 as the major resistance candidates to ceritinib. Subsequently, ceritinib treatment significantly inhibited mTORC signaling and impaired purine-pyrimidine and amino acid production pathways, suggesting a metabolic and proliferation shut-down and amino acid starvation due to targeting of the upstream receptor tyrosine kinases (RTK), which are essential for cell viability and thus not identified by CRISPR screening. Further search in the ChemBL database identified InsR and IGF1R, as RTK that was potently inhibited by ceritinib. PI-naïve and PI-resistant cell lines as well as MM patients were negative for ALK, but positive for InsR and IGF1R expression. Accordingly, co-treatment of PI-resistant cells with a combination of InsR/IGF1R inhibitors and CFZ induced strong synergistic cytotoxicity, resembling the synergistic cytotoxicity observed for ceritinib and CFZ, whereas InsR/IGF1R inhibitors did not show any synergistic cytotoxicity with ceritinib, suggesting targeting the same pathway. Importantly, the combination of ceritinib and CFZ was superior to CFZ in an orthotopic mouse model bearing PI-resistant MM cells and showed strong synergistic cytotoxicity in primary cells from MM patients progressing under or after PI-containing therapy. Conclusion: Ceritinib, an FDA-approved drug, overcomes PI resistance in MM by targeting InsR/IGF1R signaling, which is essential for PI resistance in MM. Therefore, ceritinib represents a promising, potential option for the treatment of PI-resistant MM.