Human Myeloma Research Articles

Abstract 2841: Development and validation of a systemic human multiple myeloma model utilizing luciferase expressing MM1.S tumor cells

Abstract Multiple Myeloma (MM) is a complex hematologic malignancy characterized by clonal proliferation of transformed plasma cells leading to overproduction of monoclonal immunoglobulins and subsequently end-stage organ damage. Despite therapeutic advances, MM is an incurable disease with poor prognosis in high-risk patients, and thus relevant preclinical models are necessary to develop novel treatment strategies. We developed a Multiple Myeloma in vivo model by systemic implantation of the luciferase-expressing glucocorticoid sensitive tumor cell line MM1.S (MM1.S-Luc) into immunodeficient NCG mice. First, we generated a MM1.S-Luc monoclonal cell line from a polyclonal pool of stable luciferase-expressing cells by limiting dilution. Single cells from the stable pool were expanded under selective pressure and four MM1.S-Luc monoclonal lines were selected based on in vitro growth properties and bioluminescence intensity. Next, we determined the in vivo tumor growth profile by testing the monoclonal lines at two different cell inoculums, and disease progression was monitored by in vivo serial bioimaging. MM1.S-Luc tumor growth kinetics revealed a long latency and based on our in vivo imaging results, the tumor preferentially localized to the long bones, lungs, and mandible. For validation of the MM1.S-Luc in vivo model, we determined the efficacy and survival benefit in response to various clinically relevant standard of care agents including Dexamethasone, Daratumumab, Cyclophosphamide, Vincristine, Panobinostat, and Bortezomib. The quantification of tumor burden by bioluminescent imaging showed reduced tumor burden and prolonged survival with Panobinostat and Cyclophosphamide. In summary, we have developed a systemic MM1.S-Luc model that recapitulates the disease dissemination and invasiveness of multiple myeloma for evaluating novel therapeutic approaches to treatment. Citation Format: Bincy John, Christopher Currie, Nicole Westrick, Tyler Rowe, Andrew Wong, Elizabeth Rainbolt, Zachary Ward, Chassidy Hall, David Harris. Development and validation of a systemic human multiple myeloma model utilizing luciferase expressing MM1.S tumor cells [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 2841.

Abstract 2305: Label-free high dimensional single cell morphological profiling of different hematological malignancies by ghost cytometry

Abstract Characterizing the heterogeneity in human leukemias is critical for understanding mechanisms of disease onset, uncovering biomarkers for disease diagnosis, and guiding research into novel treatment approaches. While extensive research focus has been placed on characterizing the molecular heterogeneity in leukemia by modern omics technologies, comparatively less emphasis has been placed on studying the diversity in holistic morphological changes that occur in single cells in the context of disease. Here, we report on a novel approach to understanding and characterizing different hematological malignancies using ghost cytometry, a recently developed high-content flow cytometric method that leverages high-speed, artificial intelligence (AI) driven morphological characterization and analysis of single cells. We performed morphometric profiling on human bone marrow samples from patients with T and B-cell acute lymphoblastic leukemia (ALL, n=5), acute myeloid leukemia (AML, n=5), multiple myeloma (MM, n=5) and healthy bone marrow mononuclear cell (BM-MNC) controls (n=6). Morphological profiles from individual cells were analyzed by unsupervised machine learning (universal manifold approximation and projection, UMAP) and compared within disease subsets and against controls. We identified disease-specific cell populations from a mixture of healthy control and disease samples without any labeling, based on completely label-free information. For validation of the specific cell subsets, we overlaid known cell surface markers (e.g. CD45 for blast cells, CD10/CD19 for B-ALL, CD33/CD34 for AML, CD38/CD138 for MM). The intensity of CD45 was dimmer and disease-specific markers were highly expressed in the disease-specific cell population. We also identified a unique cell subpopulation in the disease-specific cell population that could not be distinguished by existing markers. We also analyzed three human myeloma cell lines (AMO1, KMS, and L363) for comparison to the primary MM samples. In the UMAP analysis using LF-GC data, in all three types of cell lines, Bortezomib-resistant cell lines exhibited different distributions from the control parental cell lines. In addition, we observed distinct morphological profiles between primary myeloma samples and myeloma cell lines. Here we present a novel cell characterization approach for human hematological diseases that leverages AI-based, label-free, high-speed morphological characterization of single cells. We demonstrate that the approach can be used to identify subtle morphological differences in blast cells from a range of blood cancer subtypes. Application of morphological profiling and AI can be used to identify measurable, disease-related changes in these diseases that have diagnostic, drug screening, and therapeutic monitoring potential. Citation Format: Asako Tsubouchi, Juho J. Miettinen, Keisuke Wagatsuma, Satoru Akai, Yuri An, Mika Uematsu, Philipp Sergeev, Dimitrios Tsallos, Markus J. Vähä-Koskela, Sadao Ota, Caroline A. Heckman. Label-free high dimensional single cell morphological profiling of different hematological malignancies by ghost cytometry [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 2305.

Retraction: Insulin-like Growth Factor-1 Induces Adhesion and Migration in Human Multiple Myeloma Cells via Activation of β1-Integrin and Phosphatidylinositol 3'-Kinase/AKT Signaling.

Retraction: Insulin-like Growth Factor-1 Induces Adhesion and Migration in Human Multiple Myeloma Cells via Activation of β1-Integrin and Phosphatidylinositol 3'-Kinase/AKT Signaling.

Retraction: Mechanisms by which SGN-40, a Humanized Anti-CD40 Antibody, Induces Cytotoxicity in Human Multiple Myeloma Cells: Clinical Implications.

Retraction: Mechanisms by which SGN-40, a Humanized Anti-CD40 Antibody, Induces Cytotoxicity in Human Multiple Myeloma Cells: Clinical Implications.

Retraction: Cytokines Modulate Telomerase Activity in a Human Multiple Myeloma Cell Line.

Retraction: Cytokines Modulate Telomerase Activity in a Human Multiple Myeloma Cell Line.

Retraction: Effects of Oligonucleotide N3'→P5' Thio-phosphoramidate (GRN163) Targeting Telomerase RNA in Human Multiple Myeloma Cells.

Retraction: Effects of Oligonucleotide N3'→P5' Thio-phosphoramidate (GRN163) Targeting Telomerase RNA in Human Multiple Myeloma Cells.

Development of New CD38 Targeted Peptides for Cancer Imaging

PurposeMultiple myeloma (MM) affects over 35,000 patients each year in the US. There remains a need for versatile Positron Emission Tomography (PET) tracers for the detection, accurate staging, and monitoring of treatment response of MM that have optimal specificity and translational attributes. CD38 is uniformly overexpressed in MM and thus represents an ideal target to develop CD38-targeted small molecule PET radiopharmaceuticals to address these challenges.ProceduresUsing phage display peptide libraries and pioneering algorithms, we identified novel CD38 specific peptides. Imaging bioconjugates were synthesized using solid phase peptide chemistry, and systematically analyzed in vitro and in vivo in relevant MM systems.ResultsThe CD38-targeted bioconjugates were radiolabeled with copper-64 (64Cu) with100% radiochemical purity and an average specific activity of 3.3 – 6.6 MBq/nmol. The analog NODAGA-PEG4-SL022-GGS (SL022: Thr-His-Tyr-Pro-Ile-Val-Ile) had a Kd of 7.55 ± 0.291 nM and was chosen as the lead candidate. 64Cu-NODAGA-PEG4-SL022-GGS demonstrated high binding affinity to CD38 expressing human myeloma MM.1S-CBR-GFP-WT cells, which was blocked by the non-radiolabeled version of the peptide analog and anti-CD38 clinical antibodies, daratumumab and isatuximab, by 58%, 73%, and 78%, respectively. The CD38 positive MM.1S-CBR-GFP-WT cells had > 68% enhanced cellular binding when compared to MM.1S-CBR-GFP-KO cells devoid of CD38. Furthermore, our new CD38-targeted radiopharmaceutical allowed visualization of tumors located in marrow rich bones, remaining there for up to 4 h. Clearance from non-target organs occurred within 60 min. Quantitative PET data from a murine disseminated tumor model showed significantly higher accumulation in the bones of tumor-bearing animals compared to tumor-naïve animals (SUVmax 2.06 ± 0.4 versus 1.24 ± 0.4, P = 0.02). Independently, tumor uptake of the target compound was significantly higher (P = 0.003) compared to the scrambled peptide, 64Cu-NODAGA-PEG4-SL041-GGS (SL041: Thr-Tyr-His-Ile-Pro-Ile-Val). The subcutaneous MM model demonstrated significantly higher accumulation in tumors compared to muscle at 1 and 4 h after tracer administration (SUVmax 0.8 ± 0.2 and 0.14 ± 0.04, P = 0.04 at 1 h; SUVmax 0.89 ± 0.01 and 0.09 ± 0.01, P = 0.0002 at 4 h).ConclusionsThe novel CD38-targeted, radiolabeled bioconjugates were specific and allowed visualization of MM, providing a starting point for the clinical translation of such tracers for the detection of MM.

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.

Oncolytic Myxoma virus Increases Autophagy in Multiple Myeloma

Multiple myeloma, which affects plasma cells, is the second most common hematological malignancy. Despite the development of new drugs and treatment protocols, patient survival has not reached the desired level. In this study, we investigated the effects of Myxoma virus (MYXV), an oncolytic virus, on autophagy in myeloma cells. We analyzed protein expressions of ATG-5, p62, Beclin-1, LC3B, and the apoptosis marker Bcl-2 as autophagy markers in human U-266 and mouse MOPC-315 myeloma cell lines subjected to different doses of MYXV. In addition, autophagic images of myeloma cells were investigated using transmission electron microscopy (TEM). In the first 24 h, which is the early stage of autophagy, ATG-5 and Beclin-1 expression levels were increased in the U-266 and MOPC-315 cell lines in the groups that had received MYXV at a multiplicity of infection of 15. At 48 h, a significant increase was detected in the expression of LC3B, which is a late indicator. Autophagosomes were observed in myeloma cells by TEM. MYXV shows an antimyeloma effect by increasing autophagy in myeloma cells.

Deguelin inhibits the proliferation of human multiple myeloma cells by inducing apoptosis and G2/M cell cycle arrest: Involvement of Akt and p38 MAPK signalling pathway.

Deguelin exhibits antiproliferative activity against various cancer cell types. Previous studies have reported that deguelin exhibits pro-apoptotic activity against human cancer cells. The current study aimed at further elaborating the anticancer effects of deguelin against multiple myeloma cells. Cell growth estimations were made through MTT assay. Phase contrast microscopy was used for the analysis of the viability of multiple myeloma cells. Colony formation from multiple myeloma cells was studied using a clonogenic assay. Antioxidative assays for determining levels of glutathione (GSH) and superoxide dismutase (SOD) were carried out after treating multiple myeloma cells with deguelin. The apoptosis of multiple myeloma cells was studied using AO/EB and Annexin V-FITC/PI staining methods. Multiple myeloma cell cycle analysis was performed through flow cytometry. mRNA expression levels were depicted using qRT-PCR. Migration and invasion of multiple myeloma cells were determined with the wound-healing and transwell assays, respectively. Deguelin specifically inhibited the multiple myeloma cell growth while the normal plasma cells were minimally affected. Multiple myeloma cells when treated with deguelin exhibited remarkably lower viability and colony-forming ability. Multiple myeloma cells treated with deguelin produced more SOD and had higher GSH levels. The multiple myeloma cell growth, migration, and invasion were significantly declined by in vitro administration of deguelin. In conclusion, deguelin treatment, when applied in vitro, induced apoptotic cell death and resulted in mitotic cessation at the G2/M phase through modulation of cell cycle regulatory mRNAs in multiple myeloma cells.

ARK5 enhances cell survival associated with mitochondrial morphological dynamics from fusion to fission in human multiple myeloma cells

5′ adenosine monophosphate–activated protein kinase–related kinase 5 (ARK5) is involved in mitochondrial ATP production and associated with poor prognosis of multiple myeloma (MM). However, the molecular mechanisms of ARK5 in MM remain largely unknown. This study examined the pathogenic role of ARK5 in mitochondria by using genetically modified isogenic cell clones with or without ARK5 in human myeloma cell lines, KMS-11 and Sachi, which overexpress ARK5. The biallelic knockout of ARK5 (ARK5-KO) inhibited cell proliferation, colony formation, and migration with increased apoptosis. Mitochondrial fusion was enhanced in ARK5-KO cells, unlike in ARK5 wild-type (ARK5-WT) cells, which exhibited increased mitochondrial fission. Furthermore, ARK5-KO cells demonstrated a lower phosphorylated dynamin–related protein 1 at serine 616, higher protein expression of mitofusin-1 (MFN1) and MFN2, optic atrophy 1 with a lower level of ATP, and higher levels of lactate and reactive oxygen species than ARK5-WT cells. Our findings suggest that ARK5-enhanced myeloma cells can survive associated mitochondrial fission and activity. This study first revealed the relationship between ARK5 and mitochondrial morphological dynamics. Thus, our outcomes show novel aspects of mitochondrial biology of ARK5, which can afford a more advanced treatment approach for unfavorable MM expressing ARK5.

SSBP1 is a novel prognostic marker and promotes disease progression via p38MAPK signaling pathway in multiple myeloma.

Multiple myeloma (MM) remains an incurable disease. Identification of meaningful co-expressed gene clusters or representative biomarkers of MM may help to identify new pathological mechanisms and promote the development of new therapies. Here, we performed weighted sgene co-expression network analysis and a series of bioinformatics analysis to identify single stranded DNA binding protein 1 (SSBP1) as novel hub gene associated with MM development and prognosis. In vitro, CRISPR/cas9 mediated knockdown of SSBP1 can significantly inhibit the proliferation of MM cells through inducing apoptosis and cell cycle arrest in G0/G1 phase. We also found that decreased SSBP1 expression significantly increased mitochondrial reactive oxygen species (mtROS) generation and the level of phosphorylated p38MAPK. Furthermore, it was further verified that disruption of SSBP1 expression could inhibit the tumor growth via p38MAPK pathway in a human myeloma xenograft model. In summary, our study is the first to demonstrate that SSBP1 promotes MM development by regulating the p38MAPK pathway.

Production of anti-human cannabinoid receptor 2 (CB2) monoclonal antibody using non-viral vector induced human CB2 expressing myeloma as an immunogen.

Cannabinoid receptor 2 (CB2) of the cannabinoid system is predominantly expressed on immune cells and involved in a diverse range of immune functions. However, the role of CB2 in immunoregulation is still controversial. One of the challenges in the detailed characterization and functional study of CB2 is the lack of CB2-specific monoclonal antibodies (mAbs). We aimed to produce mAbs against a native form of human CB2 using human CB2 expressing mouse myeloma cells as immunogens. Non-viral vector expression system was used to generate stable human CB2-expressing mouse myeloma cells and were utilized as an immunogen for mouse immunization. Hybridoma technique was employed in the production of mAbs. The produced mAbs were verified by flow cytometry and western blotting. Using a non-viral vector expression system, myeloma clones, which stable expressed human CB2, were generated and used as immunogen for antibody production. Following mouse immunization process, the anti-CB2 polyclonal antibodies were induced. By hybridoma technique, a mAb against CB2 could be generated. This mAb reacted to CB2-expressing THP-1 cells, but not to non-CB2-expressing SH-SY5Y cells. By western blotting, the generated anti-CB2 mAb reacted with a 42 kDa protein presented in lysates of CB2-expressing THP-1 cells, but not with non-CB2-expressing SH-SY5Y cell lysates. A new approach using human CB2 expressing myeloma cells as immunogen for production of anti-CB2 mAb was developed. The generated anti-human CB2 mAb is regarded as a valuable tool for CB2 characterization. Moreover, the developed technique can be applied to produce other antibodies of interest.

Bortezomib-encapsulated metal-phenolic nanoparticles for intracellular drug delivery.

Bortezomib (BTZ) is an important boronate proteasome inhibitor that is widely used in cancer therapy. However, the clinical application of BTZ suffers from poor stability and serious adverse effects. Herein, we fabricated metal-polyphenol nanoparticles for the covalent encapsulation of BTZ. BTZ-encapsulated tannic acid (TA)-Fe3+ nanoparticles can be prepared by mixing BTZ, TA, and ferric chloride owing to the formation of metal-polyphenol coordination interaction and dynamic boronate ester bonds. The BTZ-encapsulated TA-Fe3+ nanoparticles (BTZ NPs) are stable in physiological environment (pH 7.4) with minimal drug leakage. However, BTZ NPs can be disassembled in an acidic environment. Therefore, BTZ can be rapidly released from BTZ NPs in an acidic environment (pH 5.0). More than 50% BTZ can be released from BTZ NPs after 8 h incubation at pH 5.0. BTZ NPs exhibited high cytotoxicity against human osteosarcoma Saos-2 cells and human multiple myeloma OPM-2 cells. The metal-polyphenol nanoparticles can be a promising nanoplatform for the delivery of BTZ with simultaneously enhanced therapeutic efficacy and reduced side effects.

Formononetin inhibits IgE by huPlasma/PBMCs and mast cells/basophil activation via JAK/STAT/PI3-Akt pathways.

Food allergy is a prevalent disease in the U.S., affecting nearly 30 million people. The primary management strategy for this condition is food avoidance, as limited treatment options are available. The elevation of pathologic IgE and over-reactive mast cells/basophils is a central factor in food allergy anaphylaxis. This study aims to comprehensively evaluate the potential therapeutic mechanisms of a small molecule compound called formononetin in regulating IgE and mast cell activation. In this study, we determined the inhibitory effect of formononetin on the production of human IgE from peripheral blood mononuclear cells of food-allergic patients using ELISA. We also measured formononetin's effect on preventing mast cell degranulation in RBL-2H3 and KU812 cells using beta-hexosaminidase assay. To identify potential targets of formononetin in IgE-mediated diseases, mast cell disorders, and food allergies, we utilized computational modeling to analyze mechanistic targets of formononetin from various databases, including SEA, Swiss Target Prediction, PubChem, Gene Cards, and Mala Cards. We generated a KEGG pathway, Gene Ontology, and Compound Target Pathway Disease Network using these targets. Finally, we used qRT-PCR to measure the gene expression of selected targets in KU812 and U266 cell lines. Formononetin significantly decreased IgE production in IgE-producing human myeloma cells and PBMCs from food-allergic patients in a dose-dependent manner without cytotoxicity. Formononetin decreased beta-hexosaminidase release in RBL-2H3 cells and KU812 cells. Formononetin regulates 25 targets in food allergy, 51 in IgE diseases, and 19 in mast cell diseases. KEGG pathway and gene ontology analysis of targets showed that formononetin regulated disease pathways, primary immunodeficiency, Epstein-Barr Virus, and pathways in cancer. The biological processes regulated by formononetin include B cell proliferation, differentiation, immune response, and activation processes. Compound target pathway disease network identified NFKB1, NFKBIA, STAT1, STAT3, CCND1, TP53, TYK2, and CASP8 as the top targets regulated at a high degree by formononetin. TP53, STAT3, PTPRC, IL2, and CD19 were identified as the proteins mostly targeted by formononetin. qPCR validated genes of Formononetin molecular targets of IgE regulation in U266 cells and KU812 cells. In U266 cells, formononetin was found to significantly increase the gene expression of NFKBIA, TP53, and BCL-2 while decreasing the gene expression of BTK TYK, CASP8, STAT3, CCND1, STAT1, NFKB1, IL7R. In basophils KU812 cells, formononetin significantly increased the gene expression of NFKBIA, TP53, and BCL-2 while decreasing the gene expression of BTK, TYK, CASP8, STAT3, CCND1, STAT1, NFKB1, IL7R. These findings comprehensively present formononetin's mechanisms in regulating IgE production in plasma cells and degranulation in mast cells.

EVALUATING THE EFFECT OF PRODUCTS EXTRACTED FROM PANAX BIPINNATIFIDUS SEEM ON MULTIPLE MYELOMA CELLS

Panax bipinnatifidus Seem (PB) is a precious medicinal plant in Vietnam. Although known for its many biological activities, the impact of this herb on multiple myeloma has not been elucidated. Objective: The study was conducted to evaluate the effects of total saponin extract from PB on the viability, reactive oxygen species (ROS), and mitochondrial cardiolipin content of multiple myeloma cells. Methods: Human myeloma cell line (KMS-20) cells were divided into 2 groups: control group and experimental group. In the control group, cells were grown in medium containing DMSO 0.05%. In the experimental group, cells were grown in medium containing total saponin extract (PB-TS) at different concentration ranges. Taxol is an anti-cancer drug used as a positive control in the study. Cell viability, ROS, and cardiolipin content were determined using suitable kits. Results: IC50 values of PB-TS for KMS-20 cells were 4.43±0.78 (µg/ml), higher than the IC50 value of Taxol (2.88±0.51 ng/ml). Besides, the CM-H2DCFDA fluorescence density of the cell group treated with Taxol and PB-TS increased to 121.52 ± 5.16% and 134.27 ± 3.59% compared to the control group (p<0.05). Moreover, the NAO fluorescence density of the cell group treated with Taxol and PB-TS also decreased to 62.73±6.87% and 41.56± 8.52%, respectively (p<0.05 compared with the control). Conclusion: The results of the study showed that total saponin from PB exhibited strong toxicity to KMS-20 cells through stimulation of ROS production and reduction of mitochondrial cardiolipin content.

CD46-Targeted Theranostics for PET and 225Ac-Radiopharmaceutical Therapy of Multiple Myeloma.

Multiple myeloma is a plasma cell malignancy with an unmet clinical need for improved imaging methods and therapeutics. Recently, we identified CD46 as an overexpressed therapeutic target in multiple myeloma and developed the antibody YS5, which targets a cancer-specific epitope on this protein. We further developed the CD46-targeting PET probe [89Zr]Zr-DFO-YS5 for imaging and [225Ac]Ac-DOTA-YS5 for radiopharmaceutical therapy of prostate cancer. These prior studies suggested the feasibility of the CD46 antigen as a theranostic target in multiple myeloma. Herein, we validate [89Zr]Zr-DFO-YS5 for immunoPET imaging and [225Ac]Ac-DOTA-YS5 for radiopharmaceutical therapy of multiple myeloma in murine models. In vitro saturation binding was performed using the CD46 expressing MM.1S multiple myeloma cell line. ImmunoPET imaging using [89Zr]Zr-DFO-YS5 was performed in immunodeficient (NSG) mice bearing subcutaneous and systemic multiple myeloma xenografts. For radioligand therapy, [225Ac]Ac-DOTA-YS5 was prepared, and both dose escalation and fractionated dose treatment studies were performed in mice bearing MM1.S-Luc systemic xenografts. Tumor burden was analyzed using BLI, and body weight and overall survival were recorded to assess antitumor effect and toxicity. [89Zr]Zr-DFO-YS5 demonstrated high affinity for CD46 expressing MM.1S multiple myeloma cells (Kd = 16.3 nmol/L). In vitro assays in multiple myeloma cell lines demonstrated high binding, and bioinformatics analysis of human multiple myeloma samples revealed high CD46 expression. [89Zr]Zr-DFO-YS5 PET/CT specifically detected multiple myeloma lesions in a variety of models, with low uptake in controls, including CD46 knockout (KO) mice or multiple myeloma mice using a nontargeted antibody. In the MM.1S systemic model, localization of uptake on PET imaging correlated well with the luciferase expression from tumor cells. A treatment study using [225Ac]Ac-DOTA-YS5 in the MM.1S systemic model demonstrated a clear tumor volume and survival benefit in the treated groups. Our study showed that the CD46-targeted probe [89Zr]Zr-DFO-YS5 can successfully image CD46-expressing multiple myeloma xenografts in murine models, and [225Ac]Ac-DOTA-YS5 can effectively inhibit the growth of multiple myeloma. These results demonstrate that CD46 is a promising theranostic target for multiple myeloma, with the potential for clinical translation.

A p53 score derived from TP53 CRISPR/Cas9 HMCLs predicts survival and reveals a major role of BAX in the response to BH3 mimetics

AbstractTo establish a strict p53-dependent gene-expression profile, TP53−/− clones were derived from TP53+/+ and TP53−/mut t(4;14) human myeloma cell lines (HMCLs) using CRISPR/Cas9 technology. From the 17 dysregulated genes shared between the TP53−/− clones from TP53+/+ HMCLs, we established a functional p53 score, involving 13 genes specifically downregulated upon p53 silencing. This functional score segregated clones and myeloma cell lines as well as other cancer cell lines according to their TP53 status. The score efficiently identified samples from patients with myeloma with biallelic TP53 inactivation and was predictive of overall survival in Multiple Myeloma Research Foundation–coMMpass and CASSIOPEA cohorts. At the functional level, we showed that among the 13 genes, p53-regulated BAX expression correlated with and directly affected the MCL1 BH3 mimetic S63845 sensitivity of myeloma cells by decreasing MCL1-BAX complexes. However, resistance to S63845 was overcome by combining MCL1 and BCL2 BH3 mimetics, which displayed synergistic efficacy. The combination of BH3 mimetics was effective in 97% of patient samples with or without del17p. Nevertheless, single-cell RNA sequencing analysis showed that myeloma cells surviving the combination had lower p53 score, showing that myeloma cells with higher p53 score were more sensitive to BH3 mimetics. Taken together, we established a functional p53 score that identifies myeloma cells with biallelic TP53 invalidation, demonstrated that p53-regulated BAX is critical for optimal cell response to BH3 mimetics, and showed that MCL1 and BCL2 BH3 mimetics in combination may be of greater effectiveness for patients with biallelic TP53 invalidation, for whom there is still an unmet medical need.

Small Molecule Inhibition of MAGE-A3 Induces Cell Cycle Arrest and Apoptosis in Multiple Myeloma

Background: The Type I Melanoma Antigen Gene-A3 (MAGE-A3) is commonly expressed in multiple myeloma (MM), a cancer of plasma cells. MAGE-A3 binds to Kap1 to form ubiquitin (Ub) ligase complexes and is associated with inhibition of apoptosis, resistance to chemotherapy, and cell cycle dysregulation. The highly conserved MAGE Homology Domain (MHD) undergoes a conformation change from “closed” to “open” in order to complex with Kap1, activate Ub ligase activity, and bind substrates. RNA interference (RNAi) knockdown of MAGE-A3 in MM cells results in upregulation of the endogenous CDK4/6 inhibitor P21 Cip1 and pro-apoptotic BIM, resulting in cell cycle arrest and intrinsic apoptosis. Knockdown of MAGE-A3 also increased cell death in response to melphalan and panobinostat, but not to bortezomib or cis-platin. These findings lead to the hypothesis that small molecule inhibition (SMI) of MAGE-A3 Ub ligase activity will have therapeutic application in this disease. Methods: We used the Schrodinger suite to perform in silico screening of a custom designed library of compounds using the crystal structure of the MAGE-A3 MHD to identify potential binding molecules that stabilize the closed MHD conformation. Candidates were then screened on human myeloma cell lines (HMCL) by CellTiter-Glo assay to identify compounds that reduced cell viability. Isothermal titration calorimetry was used to identify candidate SMI that bound to the MHD. HMCL were co-incubated with SMI; cell cycling was assessed by BrdU assay and apoptosis by Annexin V staining. Protein expression of p21 Cip1, BIM, and MAGE-A3 was assessed by Western blot. Combinatorial indexes in co-incubation experiments with ergotamine and chemotherapy agents were calculated with the Chou-Talalay method using CompuSyn. Results: Structure-based and biochemical screening nominated ergotamine as a candidate MAGE-A3 SMI. Ergotamine bound to the MAGE-A3 MHD and induced cell cycle arrest and apoptosis in MAGE-A3-expressing human myeloma and lymphoma cell lines and increased expression of pro-apoptotic BIM and p21 Cip1, similar to results with RNAi knockdown. These effects were not observed in negative control MAGE-A3(-) lymphoma cells. Co-incubation of ergotamine with chemotherapy agents melphalan or panobinostat resulted in synergistic killing of MAGE-A3+ HMCL. Ergotamine did not synergize with bortezomib or cis-platin under these conditions. Conclusions: These results support the combined structural and biochemical methods as an efficient means of screening for candidate inhibitors for MAGE-A3. Ergotamine recapitulated the results of RNAi knockdown of MAGE-A3 in cells that express the target, supporting a tool compound for further pharmacologic development. MAGE-A3 SMI have the potential to be a novel class of MM therapy that may be effective alone or in combination with chemotherapy or immune oncology agents. MAGE inhibitors may have broader application in other target-expressing cancers, including lung, melanoma, breast, and ovarian cancers, which are among the most common causes of cancer death.

Transgenic Mouse Model for Translational Immunotherapy Studies on Multiple Myeloma

Background Genetically engineered mouse models that recapitulate key immunological features of human multiple myeloma may lend themselves to preclinical design and testing of novel immune therapies that target members of the tumor necrosis factor (TNF) receptor superfamily in addition to BCMA. One model of this sort are double-transgenic IL6Myc mice that spontaneously develop myeloma-like plasma cell tumors (PCTs) with full genetic penetrance (100% tumor incidence) and relatively short latency (4-5 months) in presence of an intact innate and adaptive immune system (Rutsch et. al. Blood 2010). IL6Myc mice exhibit serum M-spikes, respond to proteasome inhibition, and develop osteolytic lesions mimicking myeloma bone disease (Duncan K et. al. Blood Cancer J 2013, Sun F et. al. Haematologica 2020). Here, we examined changes in the immune tumor microenvironment during PCT progression in IL6Myc mice. We found that neoplastic plasma cell development was accompanied by accumulation of inhibitory T cells, T cell exhaustion, and elevated serum levels of MIP-3α and MCP-5. A subset of aberrant plasma cells expressed the TNF receptors, TACI and BAFF-R, which, together with their ligands, APRIL and BAFF, may serve as immunotherapeutic targets in myeloma not responding to BCMA-targeted treatment (Wong DP et. al. Nat Commun 2022, Braunstein M et. al. Expert Rev Hematol 2021). Materials and methods Double-transgenic IL6Myc mice were bred by intercrossing homozygous transgenic C.iMyc ΔEμ (Duncan K et. al. Blood Cancer J 2013) and heterozygous transgenic C.IL6 mice (Kovalchuk AL. et. al. Proc Natl Acad Sci USA 2002). Age-matched IL6Myc mice were investigated at an early (5-7 weeks), intermediate (10-12 weeks) and late (14-17 weeks) stage of PCT development using flow cytometry (Cytek Aurora) and serum cytokine levels (Bio-Plex) as main research tools. Results Bone marrow (BM) plasma cells, defined as CD45-gated B220 -CD138 + cells, increased during PCT development, as one might have expected: 10.5 ± 5.8% and 26.8 ± 6.4% ( p< 0.05) in early- and late-stage mice, respectively. The proportion of B220 +CD19 + B cells fell accordingly: 29.6 ± 4.1% vs 5.5 ± 4.9% ( p< 0.0001). Consistent with the notion that the BM microenvironment of PCT-bearing mice is immunosuppressive, there was a 2-fold elevation of TGF-β expressing T cells and a 4-fold elevation of IL-10 expressing T cells in the BM when late-stage mice were compared to early-stage mice. A sizeable fraction of aberrant plasma cells expressed TACI and BAFF-R in addition to BCMA ( Figure 1, left). Spleens of IL6Myc mice undergoing tumor development contained increasing numbers of FoxP3 + regulatory T cells (8.6 ± 2.1% at early stage vs 14.0 ± 4.6% at late stage, p< 0.05) and CD11b +Ly-6G/C + myeloid-derived suppressor cells (21.2 ± 9.2% vs 51.6 ± 10.1%, p< 0.01). Exhausted T cells, detected via expression of PD-1, TIM-3, LAG-3 and TIGIT, increased in sync with PCT progression ( Figure 1, right). Elevated serum levels of inflammatory cytokines, including MIP-3α (95.3 ± 41.9 pg/ml in early-stage mice vs 514 ± 279 pg/ml in late-stage mice, p< 0.01) and MCP-5 (121 ± 43.9 pg/ml vs 284 ± 70.5 pg/ml, p< 0.05), agreed with flow-based tissue data. Conclusion Neoplastic plasma cell development in IL6Myc mice takes place in an immune suppressed tumor microenvironment similar to that promoting human myeloma. IL6Myc-driven PCTs may provide a good experimental model system for novel immunotherapeutic approaches that target TACI and BAFF-R or their ligands, APRIL and BAFF. Previous work to that end holds great promise but has been challenging thus far (Goodman DB et. al. Sci Transl Med 2022, Lee L et. al. J Immunother Cancer 2023).