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Fitness and frailty can have a significant impact on the capability of a patient to tolerate cancer treatment. However, identifying these characteristics can be challenging in the setting of cancer where a range of physical and psychological factors may affect perceived fitness, and where cancer-related health deficits may be reversible. An objective tool to distinguish effects of frailty from those of disease could significantly aid patient management and treatment selection. This research investigated a real-world population of patients with myeloma seeking to define how relative impacts of different health impacts might be detected and quantified. Consecutive patients undergoing therapy for newly diagnosed or relapsed multiple myeloma at a tertiary centre were recruited over a three-year period (n=91). Data comprised standard of care tests of myeloma or general health, together with serum samples for quantitative protein analysis using SWATH mass spectrometry. Additional quality of life (QoL) assessments was performed using standard validated tools. Data sets were tested as appropriate using standard testing, survival analysis, exploratory statistical analysis, and supervised machine learning to look for features primarily reflecting fitness frailty or myeloma disease. The patient group had characteristics that were comparable with published cohorts. Initial use of Principal Component Analysis (PCA) suggested that three clinically distinct groups could be identified: those with high myeloma disease burden, those with myeloma and a high frailty burden and those with myeloma but with minimal adverse features; the groups had distinctive patterns of survival with a significantly inferior outcome in our novel high frailty burden group. Although these groups have potential clinical utility, large clinical datasets and PCA are not well suited to prospective patient classification in a routine clinical setting. Therefore, the serum mass spectrometry was performed on patient serum, and the datasets obtained were explored to identify candidate serum protein biomarkers that could prospectively identify the groups. Supervised analysis of serum proteins using Random Forest was performed on a representative group of patients to identify potential biomarkers. This was refined to identify a candidate set using standard significance approaches ANOVA, T-test and ROC analysis as well as looking for potential biological differences between proteins in the different subsets. The QoL results were used to 10 examine the relationship between frailty, myeloma and QoL. The EORTC MY-20 questionnaire was found to be a suitable and acceptable tool. Analysis of the results of QoL data demonstrated a deterioration of QoL as the number of lines of therapy increased and was related to the overall survival in our cohort. It was also shown that our novel frail cohort of patients had an inferior self-reported quality of life. In summary, this study has identified clinically important subsets of patients that can be identified by standard of case testing and serum protein analysis. The findings of this research should require validation in prospective analyses but have the potential for changing treatment selection and management of patients with multiple myeloma.

Multiple myeloma (MM) is an incurable cancer of plasma cells, with an average five-year survival rate of approximately 50%. Over the last two decades, application of MM therapeutics, namely proteasome inhibitors (PI) and immunomodulatory imide drugs (IMiD), have almost doubled median survival time of MM patients. However, most patients will develop drug resistance. Acquired anti-cancer drug resistance remains one of the biggest barriers in the treatment of myeloma. Therefore, identifying novel therapeutics effective against MM, which are capable of overcoming drug resistance is of the utmost importance. Recently, the prolyl-tRNA synthetase (ProRS) inhibitor, halofuginone (HF), has been shown to be effective against cancer, including one study demonstrat- ing effectiveness against MM. Halofuginone is an ATP-dependent, proline and tRNA competitive inhibitor of ProRS. Recently, a new proline-uncompetitive, ATP-competitive ProRS inhibitor, NCP26, has been developed. In this work the application of HF and NCP26 in MM is explored using both MM cell lines and primary MM patient samples. HF and NCP26 demonstrated cytotoxicity to both drug-sensitive and proteasome inhibitor (PI)-resistant MM cell lines in a dose dependent manner. The anti-MM effects of HF were abrogated in the presence of excess proline, whereas excess proline had no effect on NCP26 treatment. Using bulk RNA-seq, HF and NCP26 treatment was shown to activate the amino acid starvation response (AAR), endoplasmic reticulum (ER) stress and downstream apoptotic signalling pathways in MM cell lines. NCP26 resistant MM cells also showed significant suppression of ER-associated elements, as well as reduced expression of ATF4 (the master regulator of amino acid metabolism). Single-cell RNA- seq (scRNA-seq) was employed to further examine the effects of ProRS inhibition in primary patient bone marrow samples. scRNA-seq confirmed the activation of the AAR, ER stress and apoptotic mechanisms in newly-diagnosed and relapsed MM patient cells. From differential expression and composition analyses, HF and NCP26 treatment was shown to selectively target MM cells over the majority of immune cells, including NK cells, B cells and T cells. However, HF and NCP26 were also shown to elicit apoptosis in myeloid cells. From this pre-clinical validation of ProRS inhibitors, it is clear that ProRS represents a new, promising target in MM. A number of computational methods were developed to support the experiments conducted in this work. scRNA-seq and tRNA-seq analysis pipelines were developed and their performances were benchmarked. An MM classifier was also built to be able to automate cell type classification of MM patient scRNA-seq samples. The whole bone marrow (WBM) MM scRNA-seq data from this work were used to train the MM classifier. Publically available MM WBM scRNA-seq datasets were used to evaluate the performance of the MM classifier, and it was found to be fairly accurate at labelling MM cells.

Multiple Myeloma (MM) is a haematological malignancy of terminally differentiated B cells. Despite significant advances in treatment options, development of resistance and toxic side effects are still a major clinical problem, and the disease remains incurable. This highlights the need for a deeper understanding of the pathogenesis of MM and the identification of novel therapeutic targets. Dysregulated expression of the E3 ligase HUWE1 has been reported in MM and HUWE1 has subsequently been identified as a potential therapeutic target. Recent genomic studies also uncovered HUWE1 as a novel mutational driver gene in MM, further implicating the E3 ligase in MM pathogenesis. HUWE1 is known to regulate a myriad of proteins involved in key cellular processes including DNA repair, replication and apoptosis. The aim of this study was to investigate the role of HUWE1 in replication and DNA damage response (DDR) pathways in MM. Expression of HUWE1 was demonstrated to increase in a stepwise manner across plasma cell dyscrasias, and high expression in MM patients was found to confer a shorter overall survival. Knockdown of HUWE1 using shRNA technology demonstrated significantly reduced cellular proliferation compared to the control which was underpinned by a S phase arrest. Replication proteins were identified as a novel substrate of HUWE1, which was found to catalyse the polyubiquitination of the replication protein. Further investigation found that this HUWE1-mediated ubiquitination was essential for loading of these proteins to sites of DNA damage induced by replicative stress and ultimately resulted in a DNA damage repair defect. Another area of this study focused on delineating the functional consequences of clinically relevant HUWE1 mutations which are observed in approximately 5% of MM patients. Patients witha HUWE1 mutation were observed to have significantly higher number of co-occurrent non-synonymous and somatic mutations. Analysis of a panel of MM cell lines with wild-type or mutant HUWE1, identified a similar phenotype in which HUWE1 mutant cell lines exhibited higher mutation rates as determined by the PIG-A gene mutation assay and increased incidence of micronuclei formation, two characteristics indicative of genomic instability. This loss of genome stability was determined to be underpinned by a dampened DNA repair response. This work outlines a clear role for HUWE1 in genome stability and highlights that exploitation of the replication stress response may represent a therapeutic vulnerability for MM patients with a HUWE1 mutation.

Multiple myeloma (MM) is a haematological malignancy of aberrant plasma cells in the bone marrow. Genomic instability is a hallmark of MM with most patients displaying chromosomal abnormalities. Deletions of chromosome 1p (del(1p)) are a recurrent genetic event associated with high-risk disease. Located at 1p13.2 is TRIM33, an E3 ligase and transcription co-repressor. There is increasing evidence that TRIM33 is involved in the regulation of the DNA damage response (DDR) and mitotic checkpoints, both of which are important for genome stability. In this thesis, the role of TRIM33 in MM pathogenesis was investigated, focusing on its role in the DDR, and ultimately if this role could be therapeutically exploited. Analysis of data from the publicly available CoMMpass dataset revealed around 12.3% of newly diagnosed MM patients to have a copy number loss of TRIM33, and these patients have a significantly increased frequency of chromosomal structural variants and poorer overall survival. TRIM33 shRNA knockdown was performed in two MM cell lines to investigate this loss in vitro. shTRIM33 cells exhibited increased endogenous DNA damage as shown by increased formation of 53BP1 foci and increased γH2AX expression indicating a potential DNA repair defect. Furthermore shTRIM33 cells displayed increased phosphorylation of CHK1 and KAP1 indicative of activation of ATR and ATM kinases respectively. Importantly, shTRIM33 cells were sensitized to PARP inhibitor Olaparib and ATR inhibitor VE-821, and these compounds were synergistic with standard-of-care bortezomib. RNA-seq analysis revealed massive differential expression of genes associated with immunity/inflammation in shTRIM33 cells suggesting this may contribute to, or be a consequence of, the observed DNA repair defect. To conclude, this study has identified a subgroup of MM patients with TRIM33 loss who display poor outcome and chromosomal instability, likely due to a defective DDR. In the future, PARP and ATR inhibitors may be potential therapy candidates for this high-risk subgroup.

Multiple myeloma (MM) is a haematological malignancy of plasma cells. It is caused by an uncontrolled proliferative behaviour of clonal B cells. Cancer Research UK reported around 5820 new cases each year (2% of all cases) making it the 19th most common cancer. MM remains an incurable disease with a relapsing and a remitting course. The 5-year and 10-year survival rates in England are reported to be 52.3% and 29.1% respectively. However, clinical outcomes have improved significantly in the last decade because of the increased availability of novel agents (oral and parenteral) in UK clinical practice, prescribed using one of two main strategies: fixed duration therapy (FDT) or continuous therapy (CT). These agents demonstrated their safety and efficacy in large phase 3 randomised clinical trials. Real-world data remains equally as important as clinical trials because it allows the myeloma community to understand the global variations in clinical practice, and reported outcomes of these therapies in the real-world. Advanced age, frailty and co-morbidities often exclude real-world patients from clinical trials. This created a gap in the literature about efficacy of novel therapies for those patients. A better understanding of outcomes in the real-world is, therefore, required. This introductory section presents my contribution to myeloma real-world research and to the optimisation of therapeutic outcomes of novel therapies used to treat this condition, through 6 different retrospective real-world studies and one large systematic review. KP1 of 272 bortezomib patients demonstrated that cumulative dose ≥50mg is associated with improved efficacy outcomes. KP2 of 30 carfilzomib patients demonstrated that this therapy is efficacious and has a reasonably good tolerability profile (comparable to ENDEAVOR trial). The study also demonstrated how dose attenuation should be considered early on in elderly patients or those with cardiac morbidities, to improve tolerability. KP3 of 59 DTPACE patients demonstrated that this intensive cytotoxic chemotherapy can be beneficial in extending time to next treatment (TTNT) if consolidated with an autologous stem cell transplant (ASCT). Conversely, it appears to be of less benefit in non-ASCTeligible patients, or those more heavily pre-treated. In KP4 which compared FDT (n=223) in the real-world to CT (n=253), FDT conferred inferior overall survival (OS), progression-free survival (PFS), and TTNT compared to CT. KP5 of 292 patients demonstrated that the median treatment-free interval (TFI) after 1st line therapy was 6.9 months and declined with subsequent treatment phases. The same trend was observed in the different age and co-morbidity subgroups. The large systematic review of evidence of non-standard dosing of 78 different oral anticancer agents (KP6 and KP7) identified 34 papers eligible for inclusion, of which two were myeloma studies and showed limited evidence: one for thalidomide and one for lenalidomide. In KP8 which investigated infection-morbidity in 200 elderly myeloma patients, a number of baseline clinical predictors of infective episodes were identified, such as raised baseline lactate dehydrogenase (LDH) levels, chronic obstructive pulmonary disease (COPD) and smoking history. In summary, this introductory section will describe the real-world outcomes of myeloma patients treated with a number of novel agents and using different modalities. This thesis will discuss strategies/recommendations to optimise outcomes of those therapies, and to improve tolerability.

Plasma membrane proteins are ideal therapeutic targets, being both easily accessible and involved in mediating numerous cellular processes. However, the development of therapeutic agents targeting these proteins has been hindered by the difficulties associated with their identification and characterisation. Transcriptomic approaches are unreliable and previous proteomic efforts have failed to sufficiently enrich for plasma membrane (PM) proteins or were not quantitative. Plasma membrane profiling (PMP) is a novel proteomic technique that overcomes previous limitations and enables the identification and quantification of hundreds to thousands of PM proteins across multiple samples. We have adopted this technique to characterise the whole cell surface proteome in myeloma and to identify a novel antibodydrug conjugate (ADC) target. Eight primary samples and ten human myeloma cell lines were profiled by mass spectrometry using PMP. A total of 2,077 proteins were identified with high confidence across all samples of which at least 1,319 were PM proteins. This represents a substantial improvement over other reported datasets, both in terms of total PM proteins identified and in the number of primary samples quantitated. This dataset was validated by repeat profiling and by comparing the relative PMP expression values against flow cytometry expression for six different antigens. To identify a novel antibody-drug conjugate target, proteins were ranked according to a combination of a) presence of a targetable extracellular domain b) high and ubiquitous on-tumour expression and c) low off-tumour expression. High-ranking candidates were screened for internalisation by flow cytometry and microscopy. One target, SEMA4A, which was rapidly internalised and exhibited restricted healthy tissue expression, was taken forward to test for in vitro killing activity. An anti-SEMA4A ADC induced cell death exclusively in high SEMA4A-expressing cell lines in vitro and demonstrated potent activity in an in vivo xenograft model of myeloma. We also observed that the knock-down of SEMA4A using RNA-interference causes a competitive disadvantage, suggesting that target-downregulation would not be a viable mechanism of tumour escape.

The myeloid cell lineage is a fundamental element of the immune system and it can give rise to a diverse set of terminally differentiated cells, such as macrophages or osteoclasts among many others. Mutations or misregulation of gene expression may lead to severe clinical conditions, such as arthritis, osteoporosis or cancers. Epigenetics, the regulation of gene expression and chromatin remodelling, is implicated in cell differentiation, function and disease, and hence it is a promising new area to explore in order to explain underlying cellular mechanisms. Firstly, human macrophage subtypes were studied. Chemokine (C-C motif) ligand (CCL) 1 and mannose receptor were validated to be granulocyte macrophage (GM) colony stimulating factor (CSF) induced macrophage markers, while CCL2 was specifically expressed in macrophage CSF (MCSF) macrophage population. By utilising publicly available high-throughput sequencing data, new biomarkers dehydrogenase/reductase (SDR family) member 2 and CCL26 were discovered to be MCSF-macrophage specific while guanylate binding protein 5 and apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3A were highly up-regulated in GMCSF cells. Secondly, a range of gene knock-down techniques for the myeloid cell lineage were optimised and established. Lentiviral short-hairpin RNA (shRNA) delivery methods were shown to induce an undesirable pro-inflammatory response in macrophages. Furthermore, the frequently utilised cytomegalovirus promoter for gene expression was shown to be completely silenced in macrophage populations. Locked nucleic acids were selected as a suitable alternative to shRNA knock-down and by employing this new tool it was shown that a histone demethylase lysine (K)-specific demethylase (KDM) 6B is fundamental for macrophage differentiation. Finally, a small molecule GSK-J4, a potent inhibitor of histone demethylases KDM6A, KDM6B and KDM5B specific for H3K27me3 and H3K4me3, respectively, was used to dissect epigenetic signalling in osteoclasts and multiple myeloma. In osteoclasts it was shown to act mainly by inhibiting transcriptional changes required for osteoclastogenesis when MCSF-macrophages are stimulated with Receptor Activator Of Nuclear Factor Kappa-B Ligand (RANKL), as indicated by the differential increase in H3K27me3 marks, leading to inhibition of c-Jun and potentially abolition of transcription factor AP-1, required for the transcriptional initiation of nuclear factor of activated T-cells 1 (NFATc1). In multiple myeloma cells, GSK-J4 causes a dramatic increase in expression, further supported by the build-up of global H3K4me3 marks, which results in the upregulation of the unfolded protein response pathway. In both cell systems, there is an early upregulation of metallothionein genes, which in multiple myeloma was shown to increase potentially due to rapid influx of zinc ions within the first 30 minutes, and as such may cause induction of apoptosis in multiple myeloma and may inhibit differentiation of osteoclasts.

Despite improved treatments for Non-Hodgkin’s Lymphoma (NHL) and Multiple Myeloma (MM), most patients eventually relapse and these diseases remain largely incurable. This has precipitated recent research into more clinically relevant in vitro models to enable development of more effective therapies. We have validated and standardised two in vitro primary culture models using tumour samples derived from patients with NHL, Chronic Lymphocytic Leukaemia (CLL) and MM. Several novel findings have been demonstrated. In vitro sensitivity of primary NHL cells cocultured in a CD40L model predicted clinical response to bortezomib in patients receiving the drug in a phase II trial. In vitro sensitivity correlated with CD40 expression, identifying a potential surrogate biomarker for response to bortezomib. The novel HDAC inhibitor, UCL67022 was 10-fold more potent than vorinostat in NHL and produced synergy when combined with bortezomib. UCL67022 maintained its potency in primary MM samples grown in an HS-5 stromal model. It modulated cytokine secretion resulting in downregulation of cytokine-induced signalling pathways (JAK/STAT3). A novel Hsp90 inhibitor, KW-2478 maintained activity in the HS-5 model and enhanced the activity of bortezomib and melphalan. Hsp70 was identified as a potential surrogate biomarker to monitor the combinatorial effect in future clinical trials. A highly synergistic and schedule-dependent cytotoxic effect occurred when primary MM cells were pre-treated with melphalan followed by bortezomib, with important implications for future clinical trial design. IL-6, IL-8 and VEGF levels correlated with resistance to bortezomib and melphalan and were associated with activation of JAK/STAT, MAPK and PI3K/Akt signalling pathways. Antibody neutralization of IL-6, IL-8 and VEGF resulted in restoration of drug sensitivity. We have therefore demonstrated the ability of primary culture models to predict response to chemotherapy, to identify therapeutically beneficial novel agents and to enable study of tumour microenvironmental interactions responsible for drug resistance in patients with haematological malignancies.

La falta de modelos experimentales que reflejen la heterogeneidad genética del mieloma múltiple (MM) ha obstaculizado históricamente el avance de los descubrimientos terapéuticos preclínicos. Para superar esta limitación, examinamos ratones diseñados para expresar lesiones genéticas comunes del MM encontradas en pacientes, incluyendo NF-kB, BCL2, MYC, TP53, KRAS, ciclina D1, MMSET/NSD2 y c-MAF. Estas lesiones fueron activadas en células B maduras del centro germinal mediante el alelo cγ1-cre, lugar donde se cree que se origina la enfermedad. Después de nuestro estudio, encontramos dos modelos llamados BIcγ1 y MIcγ1, los cuales desarrollaron tumores de médula ósea (MO) que cumplían con los elementos clave de la patogénesis del MM. Además, los ratones mostraron respuesta in vivo a lenalidomida y combinaciones de lenalidomida, las cuales actualmente son parte de la terapia estándar en pacientes con MM. La caracterización de ambos modelos a través de ensayos celulares, moleculares e inmunológicos reveló que la adquisición de la expresión de MYC condicionó el tiempo de progresión y, a su vez, dictó mecanismos de evasión inmunológica que remodelaron de manera diferente el microambiente de la MO. Los ratones MIcγ1 con progresión rápida impulsada por la sobreexpresión inicial de MYC, exhibieron un alto número de células T CD8+ activadas y una disminución de las células T reguladoras (Treg) inmunosupresoras en la MO, mientras que los ratones BIcγ1, con adquisición tardía de la expresión de MYC, mostraron una menor infiltración de células T CD8+ en la MO y una infiltración superior de células Treg. De manera inesperada, los ratones MIcγ1 con progresión rápida respondieron a la terapia de bloqueo de puntos de control inmunitario (ICB), mientras que el modelo de progresión lenta BIcγ1 fue completamente refractario a esta misma terapia. El estudio de las diferencias en la respuesta entre ambos modelos murinos nos llevó a la conclusión de que una alta proporción de células T CD8+ frente a células Treg en la MO predijo la respuesta a la terapia ICB. A partir de estos nuevos modelos murinos, intentamos establecer líneas celulares de MM derivadas de muestras de tumor primario, con el objetivo de utilizar un panel de líneas celulares genéticamente diversas para probar las inmunoterapias. Entre ellas, las líneas celulares MM5080 y MM8273 pudieron ser injertadas en ratones singénicos inmunocompetentes, lo cual permitió probar la inmunoterapia in vivo. En estas líneas celulares, evaluamos la hipótesis de si modular la proporción de células CD8+ /Treg podría revertir la resistencia a los ICB. Nuestros hallazgos experimentales indicaron que al aumentar la citotoxicidad de las células T CD8+ mediante la combinación de anticuerpos monoclonales (moAbs) anti-PD-1 y anti-TIGIT, o al eliminar las células T reguladoras (Treg) con un moAb anti-CD25, se revirtió la resistencia a la terapia PD-1/PD-L1, lo cual resultó en un control prolongado del MM. En conclusión, hemos generado modelos de MM similares a los encontrados en pacientes, lo cual nos permite correlacionar los rasgos genéticos e inmunológicos del MM con las respuestas a la terapia preclínica. Esto resulta fundamental para los ensayos clínicos de nuevas inmunoterapias.

Multiple Myeloma (MM) is a hematologic neoplasm caused by the clonal proliferation of aberrant plasma cells in the bone marrow (BM). Hyperdiploidy and IGH translocations are the primary genetic events in MM, each affecting approximately 50% of patients. However, there are cases where the translocation partner of IGH is still unknown. Moreover, beyond genetics, epigenomic and transcriptomic studies have uncovered the extensive chromatin activation of regulatory elements in MM, from where thousands of long non-coding RNAs (lncRNAs) are expressed. We hypothesize that these lncRNAs could be involved in translocations occurring in MM. Furthermore, the inhibition of one of these lncRNA, named SMILO, triggers MM cell death, suggesting the potential of lncRNAs as novel therapeutic targets in MM. To unravel the contribution of lncRNAs to translocations in MM, we identified fusion transcripts (FTs) from strand specific RNA-seq data in samples from different B cell subpopulations and MM patient samples. After a stringent computational filtering, we detected FTs in all studied subpopulations, implying that FTs present in healthy samples may be contributing to physiological processes. Nevertheless, FTs present in healthy samples were filtered to detect MM-specific FTs. As hypothesized, the 30% of MM-specific FTs occurred with lncRNAs (lncFTs), and interestingly, an important percentage of MM-specific FTs were transcription readthroughs (RT) between adjacent genes, therefore leading to the identification of novel RNA transcript classes with unknown functional and prognostic potential in this disease. Finally, the contribution of lncFTs to the prognosis of MM patients was assessed by multivariate survival studies performed in the CoMMpass dataset including 599 MM patient samples, demonstrating that the combination of the presence of certain lncFTs with classical genomic high-risk markers can improve the prognostic stratification of MM patients. On the other hand, we propose the generation of aptamer-siRNA chimeras as RNA-based therapies that will enable the targeted silencing of oncogenic coding and non-coding genes, including lncRNAs and FTs, specifically in the pathological plasma cells of MM. The silencing is achieved with therapeutic siRNA molecules which are delivered specifically to pathological plasma cells by MM-specific RNA aptamers. To identify the MM-specific aptamers we applied a Cell-SELEX approach, where a random RNA aptamer library is subjected to iterative binding rounds with MM cell lines to enrich sequences able to bind target cells specifically. After 10 selection rounds, we identified 10 enriched aptamer clusters, from which aptamer 1 (APT1), aptamer 1 (APT3) and aptamer 1 (APT9) were selected for validation studies. Flow cytometry and confocal microscopy studies showed that all aptamers bind preferentially and internalize into MM cell lines, besides, APT3 and APT9 were minimally toxic to MM cell lines up to 5 M, suggesting they could be used as siRNAs carriers for targeted delivery without inherent toxic effects. Likewise, we identified effective siRNAs able to inhibit both SMILO lncRNA and RRM1 coding gene with known oncogenic effects in MM, so they were selected for chimera generation with the APT3 to obtain the therapeutic silencing of these genes in the MM cell. In conclusion, this doctoral thesis aimed at elucidating the involvement and the prognostic significance of lncRNAs in fusion transcripts occurring in MM, as well as to develop RNA-based therapeutic approaches that will enable the targeted silencing of these novel transcript classes together with coding genes in a cell-specific fashion.

Advancements in therapy have improved remission and prolonged survival for patients with B cell malignancies. Unfortunately, for many challenging cancer subtypes such as mantle cell lymphoma, most patients will still relapse and die from the cancer. Chimeric antigen receptor (CAR)-T cell therapy uses genetically modified T cells expressing CAR protein to recognize and kill cancers expressing a specific antigen, such as CD19. Although CD19 CAR-T therapy has been very effective against relapsed/refractory B cell cancers, antigen escape and relapse still occur in up to 40% of patients treated with CD19 CAR-T. This work describes the creation and validation of a novel, BAFF ligand-based CAR that aims to prevent antigen escape by being able to bind multiple BAFF receptors expressed by the cancer cell, rather than one antigen alone. Using cytotoxicity assays, we demonstrate the functionality and specificity of BAFF CAR-T cells in killing several types of B cell malignancies, and we further confirm their efficacy in several mouse models of cancer. These results have helped bring BAFF CAR-T therapy to Phase I clinical trials, and we hope they prove efficacious for patients with relapsed/refractory B cell malignancies.Meanwhile, interest has rapidly increased in natural killer (NK) cell-based immunotherapies. NK cells have important roles in cancer immunosurveillance and offer advantages over T cells in clinical safety and time-to-treatment. However, NK therapies face challenges in efficacy and persistence, often due to the presence of TGF-β, a powerful immunosuppressive cytokine that is frequently elevated in cancer patients and a primary cause of NK cell dysfunction. In this work, we characterize an undiscovered role of cyclin dependent kinase 5 (Cdk5) and its coactivator p35 in NK cell cytotoxicity. Using genetic tools to modulate Cdk5/p35 kinase activity in human NK cells, as well as using NK cells from p35 knockout mice, we show that Cdk5/p35 negatively regulates NK cytotoxicity. Furthermore, we show that TGF-β induces p35 expression in NK cells, while p35 knockdown or expression of dominant negative Cdk5 resists TGF-β-mediated inhibition of NK cytotoxicity. Further understanding of these mechanisms may reveal novel strategies to enhance NK-based immunotherapies and overcome their dysfunction in cancer patients.

Certain risk factors may increase the likelihood that MM patients will experience depression. This study examined the impact of demographic variables and well-being domains as risk factors for depression. MM patients (N = 140) were recruited from support groups and online social media platforms. Participants' mean age was 67 years with a mean of 7.5 years since diagnosis. The sample was 43% male and 57% female, with 91% identifying as White. Overall, 15% of participants reported experiencing significant depression symptoms. Results indicated significant negative relationships between sexual satisfaction, physical well-being, functional well-being, financial well-being, HRQoL and depression symptoms. Additionally, there was a significant positive relationship between the experience of bone pain and depression symptoms. Female participants reported significantly more depression symptoms than male participants, although the effect size for the difference was small. Younger participants reported significantly more depression symptoms than older participants and the effect size for the difference was large. Further analyses indicated that the socioemotional elements of well-being accounted for incremental variance in depression above and beyond symptom burden. The current study provided novel information about risk factors for depression in MM patients. It clarified the impact of age and gender as risk factors for depression in this population. Together, these findings may be advantageous for clinicians as they develop strategies for the prevention, assessment, and treatment of depression in MM patients.

The treatment landscape of multiple myeloma (MM) had significantly evolved in the last decade. Treatment duration can be influenced by several disease-specific and patient-related features, and accurately predicting when a patient will require their next line of treatment is challenging. This study evaluated and compared prediction models for a second line treatment among newly diagnosed MM patients using machine learning (ML) algorithms. Non-stem cell transplant adult patients (≥ 65 years old) with MM diagnosis in a large national administrative claims database between 1/1/2013 and 8/31/2017 were identified. Health plan enrollment of 6 months prior and ≥ 1 month after index diagnosis, and ≥ 1 MM-related pharmacy claim after index diagnosis were required. Time on first treatment was assessed if a patient received a second line treatment within 12 months. Statistical models were logistic regression and Cox proportional hazards regression. ML classification models, such as logistic regression (LR), penalized regression (LASSO, ridge, and elastic net), random forests (RF), and survival-based ML models, such as random survival forests (RSF) were used to develop prediction models. Models were trained using 70% of the randomly split data. A 10-fold cross-validation repeated 3 times and down-sampling method were used for training classification models. Classification and survival-based ML model performances were evaluated using area under the curve (AUC) with confusion matrix and Harrell’s concordance error rate, respectively. This study identified 1,285 eligible MM patients, and 333 (26%) of those had a second line treatment within 12 months. Statistical logistic model had better performance (AUC= 0.75) than RF. However, RF achieved the best performance with an AUC= 0.71 (accuracy= 67%, recall= 66%, F1= 51%, and F2= 59%) among other classification models. Other classification models were comparable (AUC= 0.68-0.69, accuracy= 64%-67%, recall= 59%-61%, F1= 46%-48%, F2= 52%-55%). In survival-based models, the Cox model performed better (error rate= 0.302) than ML. The log-rank splitting approach gave the best RSF performance (error rate= 0.335). In classification methods, RF and statistical logistic models both achieved good performance. However, in survival-based methods, the Cox model had better performance than RSF. The use of these methods may be subject to the study needs.

INTRODUCTION: Multiple Myeloma (MM) is a plasma cell malignancy that causes improper production of immunoglobulins and elevated levels of monoclonal protein. Resulting morbidity is a conglomeration of symptoms due to organ failure, lytic bone disease, and hematological insufficiencies. The American Cancer Society estimates more than 35,000 patients will be diagnosed with multiple myeloma in the United States in 2023. Current therapeutic regimen hinge on the idea of myeloma as a chronic disease that cannot be entirely cured and toxic chemotherapies with long-term treatment cycles are the standard of care. The need for a one-time therapy that is both safe and efficacious and with potentially curative action has led to the development of anti-BCMA CAR T-cell infusions. The overwhelming success of this novel therapy in MM has been demonstrated in clinical trials, but the need for data surrounding the long-term toxicities post-CAR T-cell treatment in a real-world population of MM patients still exists. Common expected adverse events that have been identified in clinical trials include cytokine release syndrome, neurotoxic events, hematological toxicities, and infections associated with immunosuppression. This study was formed to elucidate the long-term adverse events associated with anti-BCMA CAR T-cell therapy in a real-world patient population. METHODS: A total of 54 patients who received a CAR T-cell infusion for their relapsed and refractory multiple myeloma were studied in a retrospective analysis at Dana-Farber Cancer Institute. Data were collected prior, during, and after infusion to gauge treatment performance and toxic side effects. Analyses of collected data, including complete blood counts, serum protein electrophoresis, fluorescence in-situ hybridization (FISH) data from bone marrow biopsy, and imaging were performed. RESULTS: Patients were followed for a mean average of 165 days (range 29-462) post-infusion. Patients either received CiltaCel (n = 7) or IdeCel (n = 47). Grade 3 or greater cytopenia occurred in 48% of patients at some point following infusion and the median time to first onset was 30 days (10-189). Forty-six patients (85%) achieved a partial response or better as their best response to therapy. During inpatient infusion, 76% of patients experienced grade 1 or 2 cytokine release syndrome (CRS) and 8% experienced grade 1 or 2 immune effector cell-associated neurotoxicity syndrome (ICANS). A total of 12 patients (22%) developed infections after infusion with respiratory infections being the most frequent (17%). Nine patients were also evaluated on a closer scale for their experience with prolonged cytopenia, but no significant commonalities were found. DISCUSSION: The analysis of this study found this patient population to have a considerably less frequent incidence of high grade cytopenia as compared to clinical trial data. However, 92% of patients developed grade 1-3 anemia and 77% developed any grade thrombocytopenia, both figures are greater than those presented in the KarMMa-2 clinical trial study for ide-cel. Patients who developed severe cytopenia were able to recover absolute neutrophil counts (ANC) over the course of their follow-up appointments which is an important aspect in the prevention and avoidance of serious infection. This same recovery was not observed in platelet or hemoglobin counts. Additionally, 15 patients were reported to still have high-grade cytopenia at 30—60-days post infusion, but this number drops to only 5 patients for the 60—90-day timeframe, this steep drop is indicative of an early onset of severe cytopenia that may not carry on as the patient progresses further from their infusion date. Compared to the KarMMa-2 study which reported an infection incidence of 69%, observations from this current study suggest this real-world patient population remained healthier after infusion in terms of infection with only 23% of patients developing post-infusion infection. Instances of CRS and ICANS were comparable to data evaluated in clinical trials. Finally, treatment responses did not significantly differ between the population of patients who developed grade 3 or greater cytopenia and those patients who did not. More data is required to determine the risk-benefit profile of early intervention with CAR T-cell therapy as directly compared to the current standard of care. This study is an encouraging insight into the performance of real-world RRMM patients that should assure patients and clinicians of the safety and uncompromising efficacy of anti-BCMA therapy as a treatment option for multiple myeloma.

The tumor microenvironment (TME) has emerged as a key contributor to cancer progression in numerous solid tumors as well as blood cancers that evolve in the bone marrow (BM). Within the TME, tumor-associated macrophages (TAMs) play a significant role in cancer cell survival and progression and have been implicated in producing an immuno-suppressive TME by generating inflammatory mediators, growth factors, cytokines, chemokines, etc. In multiple myeloma (MM), a fatal plasma cell neoplasm, inflammatory and anti-viral pathways promote disease progression. Interferon regulatory factor 4 (IRF4) is a transcription factor that has previously been shown to promote MM progression through enhanced survival of malignant plasma cells. Under physiological conditions, IRF4 also regulates macrophage polarization, where its expression drives alternative activation of macrophages primarily to M2-like (anti-inflammatory) phenotypes. However, the extent to which IRF4 activation or direct inhibition governs the polarization status and pro-tumoral activity of macrophages in the MM microenvironment has not been fully elucidated. Here, we show that IRF4 activation in MM macrophage models correspond with a more M2-like phenotype and direct inhibition of IRF4 shifts the polarization status toward a more M1-like, anti-tumoral phenotype.

INTRODUCTION: Multiple myeloma (MM) is a disease that results in the production of ineffective immunoglobulins and monoclonal proteins in the blood and urine, leading to insufficient organ function or death. Currently, there is a 5-year survival rate of 47% for patients diagnosed with MM, with a proportion of patients ultimately succumbing to the disease. The current standard of care for MM includes toxic combinations of chemotherapy. The evolution of chimeric antigen receptor (CAR) T-cell therapy for hematologic cancers such as lymphoma, leukemia, and now myeloma has provided another effective treatment option for patients who have relapsed after standard treatments for MM. Idecabtagene Vicleucel (ide-cel), was approved in March 2021 for patients with relapsed and refractory MM. While CAR T-cell treatment appears to be far less toxic than standard chemotherapy, this therapy comes with its own associated toxicities, mainly cytokine release syndrome (CRS) and neurotoxicity (NT). In clinical trials, ide-cel demonstrated to be an effective treatment in some patients, leading to the FDA approval for patients who have exhausted multiple other lines of therapy. Currently, it is unclear why patients respond differently to CAR T-cell treatment and why some patients present with more severe toxicity than others. Therefore, this study aims to examine patient factors such as demographics, age, and treatment history to determine if such characteristics may influence the CAR T-cell response; also, we assess the efficacy of ide-cel in a real-world experience outside of a clinical trial. METHODS: In this study, 14 patients’ medical records were reviewed after receiving commercial CAR T-cell therapy between August 2021 and January 2022. Eligible patients for the therapy were determined by strict inclusion criteria, including having a confirmed diagnosis of MM and exhausting at least four prior lines of therapy, as well as exclusion criteria, such as excluding individuals who have received CAR T-cells prior in a clinical trial setting. Approximately one month before preparation lymphodepletion chemotherapy, eligible patients underwent leukapheresis and had their blood sent to a laboratory to extract T-cells and genetically modify them to express the CAR for reinfusion. On 3 and 5 days prior to CAR T-cell infusion, patients underwent lymphodepletion using fludarabine and cyclophosphamide. Patients remained in the hospital for approximately one week following infusion, pending adverse reactions. After discharge, patients returned to the hospital for routine follow-ups. Data analysis was then performed on collected clinical readouts such as: prior treatments, bone marrow biopsies, response rates, laboratory values from blood samples, and pre- and post-infusion scans of various tissues within the body. RESULTS: At a median follow-up time of 15 weeks, six patients (43%) achieved a complete response (CR), three patients demonstrated a partial response (PR, 21%), and four patients showed disease progression (PD, 28%). Post-infusion scans were not available for one subject (7%) as they were still in the hospital. These results are similar to the phase I and phase II trials in which 45% and 33% of patients demonstrated a CR post-infusion, respectively. As for associated toxicities, 10 patients (71%) experienced CRS and one patient (7%) presented with ICANS. All patients that achieved a CR experienced ide-cel related toxicities, compared with only 38% of those with less favorable or unknown outcomes, which indicates that systemic immune system activation which causes CRS may be required to achieve a CR but CRS is not always linked with a CR outcome. There were 28 different chemotherapy regimens used as the standard of care treatment prior to ide-cel therapy. We assessed the most recent chemotherapeutic regimen in each patient to assess whether there is an association with most recent treatment and response. Of the six patients that achieved a CR to ide-cel, all were previously treated with RVD or CyBorD regimens, compared to the four patients who had disease progression who were mainly treated with salvage DCEP chemotherapy. Four patients (29%) received DCEP as their final chemotherapy regimen, and 3 of these 4 (75%) demonstrated progressive disease after ide-cel. Two patients received Belantamab-Mafodotin prior to ide-cel treatment, with one patient presenting with disease progression and the other patient achieving CR. 71% of patients experienced CRS following ide-cel infusion, which is resembles the phase II trial of ide-cel in which 84% of patients demonstrated CRS. In this study, only 7% of patients experienced neurological toxicity, which is comparable to the 18% of patients that demonstrated to have ICANS in the phase II study. CONCLUSIONS: We found similar performance of the ide-cel CAR-T therapy in the real world setting as in the clinical trial. Also, the complete responses were achieved by subjects with an array of characteristics, including varying recent chemotherapeutic treatments, IgG, IgA, and light-chain only subtypes of MM, and diverse demographics and other characteristics. The characteristic that demonstrated the most predictability and somewhat unique to subjects with CR was the associated toxicities from ide-cel. Development of these associated toxicities may attest that substantial immune activation, of CAR T-cells and other immune cells, leads to the efficacy of the product in eliminating cancer cells. Further analysis will need to be completed as more individuals enroll in this study to be able to determine if there are significant associations between demographics and prior lines of treatment with response to ide-cel CAR-T therapy. Lastly, future studies should assess the immune cell effector functions that are generated in CR patients that will help to specify the association between ide-cel activation, experienced associated toxicities, and its efficacy.

Multiple myeloma (MM) is characterised by an expansion of malignant plasma cells in the bone marrow, systemic bone loss and destructive osteolytic bone lesions. These are mediated by an imbalance in bone remodelling, in which bone resorption is exacerbated and bone formation is suppressed. More than 90% of MM patients present with osteolytic lesions that can lead to pain and increased risk of fracture, significantly impacting quality of life. Bone-targeted treatments currently used in the clinic can suppress lesion progression and reduce fracture risk, however these agents cannot replace lost bone and patients continue to fracture. The canonical Wnt/beta-catenin signaling pathway is a potent regulator of bone formation. We sought to examine whether Wnt-targeted agents could be of therapeutic benefit to rebuild lost bone and improve bone strength in MM patients. Utilising the 5TGM1eGFP murine model of myeloma, this thesis first demonstrated that a novel anti-LRP6 antibody, which potentiates LRP6-mediated Wnt1 class signaling, prevented myeloma-induced bone disease primarily through reduced bone resorption. The greatest protection against myeloma-induced bone loss however was shown when anti-LRP6 was used in combination with the bone anabolic agent, anti-DKK1, in the 5TGM1eGFP murine model of myeloma; compared to anti-LRP6 single treatment approaches. Importantly, stimulation of the Wnt/beta-catenin signaling pathway through these therapeutic strategies does not exacerbate tumour growth, which was a concern raised previously in the literature. Moreover, this thesis established in vivo murine models of bone repair following myeloma-induced bone destruction utilising the 5T2MM and Vk*Myc cell lines. Improving upon bone repair methods published recently, this thesis provides the first evidence that anti-SOST antibody treatment could repair osteolytic lesions within the 5T2MM model of myeloma. Importantly, this thesis is the first to characterise the skeletal destruction and osteolytic lesions associated with the Vk*Myc murine model of myeloma. Results arising from this thesis will define novel therapeutic strategies that could be beneficial to address the skeletal complications associated with MM. Murine models of bone repair established in this investigation will provide opportunities for the field to explore the potential for these Wnt-targeted agents to repair bone following cancer-induced bone destruction.

Multiple myeloma (MM) is the second most common haematological malignancy and remains incurable despite therapeutic advances. 18F-FDG (FDG) PET/CT is a relevant tool MM for staging and it is the reference imaging technique for treatment evaluation. However, it has limitations, and investigation of other PET tracers is required. Preliminary results with L-methyl-[11C]- methionine (MET), suggest higher sensitivity than 18F-FDG. This study aimed to compare the diagnostic accuracy and prognostic value of 1FDG and MET in MM patients. We prospectively compared FDG and MET PET/CT for assessment of bone disease and extramedullary disease (EMD) in a series of 52 consecutive patients (8 smoldering MM, 18 newly diagnosed MM and 26 relapsed MM patients). Bone marrow (BM) uptake patterns and the detection of focal lesions (FLs) and EMD were compared. Furthermore, FDG PET parameters with known MM prognostic value were explored for both tracers, as well as total lesion MET uptake (TLMU). Median patient age was 61 years (range, 37-83 years), 54% were male, 13% of them were in stage ISS (International Staging System) III, and 31% had high-risk cytogenetics. FDG PET/CT did not detect active disease in 6 patients, while they were shown to be positive by MET PET/CT. Additionally, MET PET/CT identified a higher number of FLs than FDG in more than half of the patients (63%). For prognostication we focussed on the relapsed cohort, due to the low number of progressions in the two other cohorts. Upon using FDG PET/CT in relapsed patients, the presence of more than 3 FLs (HR 4.61, p = 0.056), more than 10 FLs (HR 5.65, p = 0.013), total metabolic tumor volume (TMTV) p50 (HR 4.91, p = 0.049) or TMTV p75 (HR 5.32, p = 0.016) were associated with adverse prognosis. In MET PET/CT analysis, TMTV p50 (HR 4.71, p = 0.056), TMTV p75 (HR 6.27, p = 0.007), TLMU p50 (HR 8.8, p = 0.04) and TLMU p75 (HR 6.3, p = 0.007) adversely affected PFS. This study confirmed the diagnostic and prognostic value of FDG in MM. In addition, it highlights that MET has higher sensitivity than FDG PET/CT for detection of myeloma lesions, including FLs. Moreover, we show, for the first time, the prognostic value of TMTV and TLMU MET PET/CT in the imaging evaluation of MM patients.

Simple Summary Multiple Myeloma (MM) remains incurable due to high relapse rates and fast development of drug resistances. Monoclonal antibodies (mAb) have revolutionized MM treatment, opening the door to chemotherapy-free yet curative treatments. Nevertheless, antibody-based therapies face several difficulties which could be overcome by nucleic acid aptamers. Aptamers are short oligonucleotide ligands that bind their targets with great affinity and specificity, and can be easily conjugated to different cargoes for their cell-specific delivery. This review summarizes the aptamers that have been tested in MM so far with promising results, and proposes various strategies for the further development of aptamer-based strategies against MM. Multiple Myeloma (MM) remains an incurable disease due to high relapse rates and fast development of drug resistances. The introduction of monoclonal antibodies (mAb) has caused a paradigm shift in MM treatment, paving the way for targeted approaches with increased efficacy and reduced toxicities. Nevertheless, antibody-based therapies face several difficulties such as high immunogenicity, high production costs and limited conjugation capacity, which we believe could be overcome by the introduction of nucleic acid aptamers. Similar to antibodies, aptamers can bind to their targets with great affinity and specificity. However, their chemical nature reduces their immunogenicity and production costs, while it enables their conjugation to a wide variety of cargoes for their use as delivery agents. In this review, we summarize several aptamers that have been tested against MM specific targets with promising results, establishing the rationale for the further development of aptamer-based strategies against MM. In this direction, we believe that the study of novel plasma cell surface markers, the development of intracellular aptamers and further research on aptamers as building blocks for complex nanomedicines will lead to the generation of next-generation targeted approaches that will undoubtedly contribute to improve the management and life quality of MM patients.

The bone marrow (BM) microenvironment acts as a breeding ground for drug resistance in multiple myeloma (MM). The interaction with bone marrow stromal cells (BMSCs) confer environment-mediated drug resistance (EMDR) to multiple myeloma. We investigated BM stroma-induced transcriptome signatures of MM cells through a sophisticated analysis of gene expression. In particular, we defined transcription program modulated by JunB, an emerging regulator of MM pathogenesis and a member of the transcription factor superfamily activator protein 1 (AP-1), in response to BM stimulation. The data and results lay down a foundation for future studies to illustrate the regulatory role of JunB in the de novo drug resistance of multiple myeloma.

Simple Summary B-cell regeneration during therapy has been associated with the outcome of multiple myeloma (MM) patients. However, the effects of therapy and hemodilution in bone marrow (BM) B-cell recovery have not been systematically evaluated. Here, we show that hemodilution is present in a significant fraction of MM BM samples, leading to lower total B-cell, B-cell precursor (BCP), and normal plasma cell (nPC) counts. Among MM BM samples, decreased percentages (vs. healthy donors) of BCP, transitional/naive B-cell (TBC/NBC) and nPC populations were observed at diagnosis. BM BCP, but not TBC/NBC, increased after induction therapy. At day+100 post-autolo-gous stem cell transplantation, a greater increase in BCP with recovered TBC/NBC numbers but persistently low memory B-cell and nPC counts were found. At the end of therapy, complete response (CR) BM samples showed higher CD19(-) nPC counts vs. non-CR specimens with no clear association between BM B-cell regeneration profiles and patient outcomes. B-cell regeneration during therapy has been considered as a strong prognostic factor in multiple myeloma (MM). However, the effects of therapy and hemodilution in bone marrow (BM) B-cell recovery have not been systematically evaluated during follow-up. MM (n = 177) and adult (>= 50y) healthy donor (HD; n = 14) BM samples were studied by next-generation flow (NGF) to simultaneously assess measurable residual disease (MRD) and residual normal B-cell populations. BM hemodilution was detected in 41 out of 177 (23%) patient samples, leading to lower total B-cell, B-cell precursor (BCP) and normal plasma cell (nPC) counts. Among MM BM, decreased percentages (vs. HD) of BCP, transitional/naive B-cell (TBC/NBC) and nPC populations were observed at diagnosis. BM BCP increased after induction therapy, whereas TBC/NBC counts remained abnormally low. At day+100 postautologous stem cell transplantation, a greater increase in BCP with recovered TBC/NBC cell numbers but persistently low memory B-cell and nPC counts were found. At the end of therapy, complete response (CR) BM samples showed higher CD19(-) nPC counts vs. non-CR specimens. MRD positivity was associated with higher BCP and nPC percentages. Hemodilution showed a negative impact on BM B-cell distribution. Different BM B-cell regeneration profiles are present in MM at diagnosis and after therapy with no significant association with patient outcome.

Although the majority of patients with myeloma who achieve undetectable minimal residual disease show prolonged survival, some of them relapse shortly afterwards. False-negative results due to hemodiluted bone marrow samples could explain this inconsistency, but there is no guidance on how to evaluate them. We analyzed three cell populations normally absent in peripheral blood in 1404 aspirates obtained in numerous disease settings and in 85 healthy adults. Pairwise comparisons according to age and treatment showed significant variability, thus suggesting that hemodilution should be preferably evaluated with references obtained after receiving identical regimens. Leveraging the minimal residual disease results from 118 patients, we showed that a comparison with age-matched healthy adults could also inform on potential hemodilution. Our study supports the routine assessment of bone marrow cellularity to evaluate hemodilution, using as reference values either treatment-specific or from healthy adults if the former are unavailable.

Purpose: To investigate within phantoms the minimum CT dose allowed for accurate attenuation correction of PET data and to quantify the effective dose reduction when a CT for this purpose is incorporated in the clinical setting. Methods: The NEMA image quality phantom was scanned within a large parallelepiped container. Twenty-one different CT images were acquired to correct attenuation of PET raw data. Radiation dose and image quality were evaluated. Thirty-one patients with proven multiple myeloma who underwent a dual tracer PET/CT scan were retrospec- tively reviewed. 18F-fluorodeoxyglucose PET/CT included a diagnostic whole-body low dose CT (WBLDCT: 120 kV-80mAs) and 11C-Methionine PET/CT included a whole-body ultra-low dose CT (WBULDCT) for attenuation correction (100 kV-40mAs). Effective dose and image quality were analysed. Results: Only the two lowest radiation dose conditions (80 kV-20mAs and 80 kV-10mAs) produced artifacts in CT images that degraded corrected PET images. For all the other conditions (CTDIvol ≥ 0.43 mGy), PET contrast recovery coefficients varied less than ± 1.2%. Patients received a median dose of 6.4 mSv from diagnostic CT and 2.1 mSv from the attenuation correction CT. Despite the worse image quality of this CT, 94.8% of bone lesions were identifiable. Conclusion: Phantom experiments showed that an ultra-low dose CT can be implemented in PET/CT procedures without any noticeable degradation in the attenuation corrected PET scan. The replacement of the standard CT for this ultra-low dose CT in clinical PET/CT scans involves a significant radiation dose reduction

Despite significant advances in the treatment of multiple myeloma which had led to unprecedented rates of response and survival, patients still relapse, and cure remains elusive. We propose in this review a roadmap to achieve the dream of cure for multiple myeloma based on five complementary strategies. First, to increase knowledge about disease pathogenesis with a focus on the biology of circulating tumor cells, responsible for dissemination and extramedullary disease, and minimal residual disease clones who represent the reservoir of clonal evolution and disease recurrence. Second, to consider undetectable measurable residual disease (MRD), defined by high-sensitive techniques, as the new endpoint of therapy. Third, to treat disease causation instead of symptomatology through early detection and intervention. Thereby, by treating high-risk smoldering myeloma patients early, we may not only contribute to delay disease progression into active disease but also to increase the cure rates. Fourth, to use the most active scheme in standard-risk patients if the cure is in the horizon. Fifth, to investigate experimental therapies in newly diagnosed patients with high-risk MM, implementing early rescue intervention strategies with the goal of eradicating all tumor clones, and achieving minimal residual disease negativity.

Multiple myeloma (MM) is a hematological malignancy characterized by the neoplastic proliferation of the plasma cells. MM is a relatively rare cancer that accounts for about 1.8% of all cancers and is the second most common hematologic malignancies, and despite the advancement from untreatable to treatable malignancy, it is yet incurable. Calcium ions (Ca2+) play an important role as second messengers in regulating a plethora of physiological and pathological processes, hence cytoplasmic Ca2+ is tightly regulated with strict spatial and temporal control to initiate, maintain, and terminate appropriate signaling pathways and phenotypes including cellular proliferation, cell cycle control, migration, gene expression, muscle contraction, metabolism, and cell death. Multiple Ca2+ pumps and channels exist in the cell to tightly regulate cytoplasmic Ca2+ levels. The plasma membrane Ca2+ transport ATPase (PMCA), and the Na+/Ca2+ exchanger (NCX) are present on the plasma membrane, the mitochondrial Ca2+ uniporter (MCU) are located on the mitochondrial membrane, and the sarcoplasmic reticulum/endoplasmic reticulum Ca2+-ATPase (SERCA) pump, the 1,4,5-triphosphate receptor (IP3R) and ryanodine receptor (RyR) channels are expressed on SE/ER membranes, all these channels work seamlessly to regulate and redistribute Ca2+ levels among various cellular compartments. The major Ca2+ regulatory pathway in non-excitable cells is the Store-Operated Ca2+ Entry Pathway (SOCE) and one its major contributors is TRPC1. TRPC1 is a member of the TRP protein superfamily and a potential modulator of store-operated Ca2+ entry (SOCE) pathways. While TRPC1 is ubiquitously expressed in most tissues, its dysregulated activity may contribute to the hallmarks of various types of cancers, including breast cancer, pancreatic cancer, glioblastoma multiforme, lung cancer, hepatic cancer, multiple myeloma and thyroid cancer. Our group has developed a novel cyclic peptide referred to as MTI-101 for the treatment of MM. MTI-101 is derived from a 10 D-amino acids peptide HYD1, which was discovered to block cancer cell adhesion to extracellular matrix. It was previously reported that acquired resistance to HYD-1 correlated with repression of genes involved in store operated Ca2+ entry (SOCE): PLCβ, SERCA, ITPR3, and TRPC1 expression. We sought to determine the role of TRPC1 heteromers in mediating MTI-101 induced cationic flux. Our data indicate that consistent with activation of TRPC heteromers, MTI-101 treatment induced Ca2+ and Na+ influx. However, replacing extracellular Na+ with NMDG did not reduce MTI-101-induced cell death. In contrast, decreasing extracellular Ca2+ reduced both MTI-101-induced Ca2+ influx as well as cell death. The causative role of TRPC heteromers was established by suppressing STIM1, TRPC1, TRPC4 or TRPC5 function both pharmacologically and by siRNA, resulting in a reduction in MTI-101-induced Ca2+ influx. Mechanistically MTI-101 treatment induces trafficking of TRPC1 to the membrane and co-immunoprecipitation studies indicate that MTI-101 treatment induces a TRPC1-STIM1 complex. Moreover, treatment with calpeptin inhibited MTI-101-induced Ca2+ influx and cell death indicating a role of calpain in the mechanism of MTI-101-induced cytotoxicity. Finally, components of the SOCE pathway were found to be poor prognostic indicators among MM patients, suggesting that this pathway is attractive for the treatment of MM.

Simple Summary Multiple myeloma (MM), the second most common hematological neoplasm, is still considered an incurable disease. Long non-coding RNAs (lncRNAs), genes that do not encode proteins, participate in numerous biological processes, but their deregulation, like that of coding genes, can contribute to carcinogenesis. Increasing evidence points to the relevant role of lncRNAs in the development of human tumors, such that they emerge as attractive biomarkers and therapeutic targets for cancer treatment, including MM. Here we review the oncogenic or tumor-suppressor functions of lncRNAs in MM and provide an overview of novel therapeutic approaches based on lncRNAs that will help to improve the management of these patients. MM is a hematological neoplasm that is still considered an incurable disease. Besides established genetic alterations, recent studies have shown that MM pathogenesis is also characterized by epigenetic aberrations, such as the gain of de novo active chromatin marks in promoter and enhancer regions and extensive DNA hypomethylation of intergenic regions, highlighting the relevance of these non-coding genomic regions. A recent study described how long non-coding RNAs (lncRNAs) correspond to 82% of the MM transcriptome and an increasing number of studies have demonstrated the importance of deregulation of lncRNAs in MM. In this review we focus on the deregulated lncRNAs in MM, including their biological or functional mechanisms, their role as biomarkers to improve the prognosis and monitoring of MM patients, and their participation in drug resistance. Furthermore, we also discuss the evidence supporting the role of lncRNAs as therapeutic targets through different novel RNA-based strategies.

Abstract: Historically, the treatment of multiple myeloma in transplant eligible patients has included induction chemotherapy followed by autologous stem cell transplant (ASCT) and a watch and wait approach until relapse. The introduction of maintenance chemotherapy has changed the traditional observation period into active treatment time. We examined the impact of lenalidomide maintenance on survival outcomes in the front line and relapsed setting. Our population included patients treated with bortezomib based induction followed by ASCT who went on to receive lenalidomide maintenance or no maintenance. Patient data was taken from the Myeloma Canada Research Network Canadian Multiple Myeloma Database (MCRN CMM-DB) which includes data from 13 academic cancer centers across Canada.Our data demonstrates that lenalidomide maintenance is associated with improved progression free (PFS) and overall survival (OS) as well as higher rates of favourable responses. We did not observe any unanticipated adverse effects. Rates of discontinuation for reasons other than relapse were low. Analysis of relapsed patients demonstrates that lenalidomide maintenance did not results in worse second PFS (time from second line therapy to second relapse, death or last follow-up) in patients who received the drug again in second line therapy. Lenalidomide maintenance was associated with improve overall survival from time of initiation of relapse therapy. Additionally, lenalidomide maintenance did not negatively impact second PFS, OS from time of initiation of relapse therapy, and rates of favourable response when lenalidomide used again in second line treatment. Our data is the first analysis of transplant eligible patients from the MCRN CMM-DB. Our data supports the continued use of lenalidomide maintenance as standard of care following ASCT. Importantly, our data suggests that the use of lenalidomide maintenance does not negatively impact survival or response outcomes when used again in second line therapy. As such, use of or relapse on lenalidomide maintenance should not be considered an exclusion criteria for use of lenalidomide in second line therapy.

Multiple myeloma (MM) is a clonal plasma B-cell neoplasm characterised by the presence of uncontrolled proliferation of antibody-secreting plasma cells in the bone marrow. Despite therapeutic advancements, MM remains incurable due to its low median survival rate and nearly all patients will eventually relapse regardless of the frontline regimen they receive. Thus, alternative therapeutic strategies that target the pathogenic and resistance mechanisms of MM need to be developed to improve overall survival in MM patients. Cancer cells generally have higher metabolic demands due to their high proliferative nature and as such, production of intracellular by-products including free reactive oxygen species (ROS) are also increased, which can cause oxidative cell death. Hence, cancer cells exploit antioxidant molecules to maintain intracellular redox homeostasis and to prevent further cell damage from the anticancer drugs that induce oxidative stress. One of the major antioxidant systems is the thioredoxin (Trx) system, which is comprised of thioredoxin (Trx), thioredoxin reductase (TrxR), and nicotinamide adenine dinucleotide phosphate (NADPH), all of which as one can scavenge ROS, reduce oxidised proteins and redox-sensitive molecules, and regulate transcription factors. Myeloma cells have been shown to have high Trx and TrxR expression, which correlates with increased cell proliferation, evasion of programmed cell death and chemoresistance. Gold compounds have a high affinity for thiol and selenol groups, which makes the Trx system vulnerable to these compounds. In this study, TrxR was targeted in myeloma cells with gold(I) compounds, including auranofin and [Au(d2pype)2]Cl. [Au(d2pype)2]Cl exhibited significant inhibition on TrxR activity in both in vitro and in vivo models, which led to induction of ROSmediated cell death in myeloma cells. [Au(d2pype)2]Cl also completely abrogated the tumourigenic capacity of myeloma cells in clonogenic assays, whereas auranofin was less effective. The expression of the MYC oncogene, which drives myeloma progression, was also downregulated in both in vitro and in vivo models when treated with [Au(d2pype)2]Cl. Auranofin and [Au(d2pype)2]Cl were further evaluated in 2D co-culture assays to determine their efficacy in overcoming environment mediated drug resistance (EMDR) in myeloma cells that are co-cultured with bone marrow stromal cells (BMSCs) in normoxic (20% O2) and hypoxic (1% O2) conditions. [Au(d2pype)2]Cl generally displayed much better efficacy by exhibiting lower IC50 values as compared to auranofin when used on either the attached or non-attached myeloma cells in both normoxia and hypoxia. Based on the potency that [Au(d2pype)2]Cl displayed over auranofin in these co-culture assays, [Au(d2pype)2]Cl was selected as a tool for subsequent studies on the role of the Trx system in the molecular mechanisms that contribute to drug resistance in MM. Cell adhesion mediated drug resistance (CAM-DR) occurs from the intercellular adhesion between myeloma cells and the bone marrow microenvironment (BMME). Several studies have indicated CAM-DR is associated with the activation of NF-ĸB and causes a subset of myeloma cells attached to BMSCs and/or extracellular matrix (ECM) to be chemo-resistant prior to MM therapy. In this study, inhibition of TrxR with [Au(d2pype)2]Cl decreased the adhesive capabilities of myeloma cells to BMSCs and was accompanied by downregulation of integrin Very Late Activation Antigen-4 (VLA-4), which is an important cell adhesion molecule responsible for CAM-DR in MM. [Au(d2pype)2]Cl treatment also significantly downregulated protein levels of the p65 subunit of NF-ĸB as well as expression of NF-ĸB-regulated genes. Moreover, the expression of VLA-4 was markedly decreased when NF-ĸB was inhibited by BAY 11-7082, inhibitor of IĸB kinase (IKK), similar to results obtained when [Au(d2pype)2]Cl was used. These findings suggest that the Trx system may affect the expression of VLA-4 in myeloma cells by regulating the NF-ĸB signalling pathway. Based on the results obtained with VLA-4, the role of another cell adhesion molecule, mucin 1 (MUC1) was evaluated in this study. MUC1 is aberrantly expressed in MM and is involved in oncogenesis as well as regulation of ROS. Since there is a possible link between the redox cellular balance and MUC1, this study investigated if inhibition of TrxR could affect MUC1 expression in myeloma cells. Results showed that inhibition of TrxR decreased the expression of MUC1 in myeloma cells that were co-cultured with BMSCs. Moreover, the adhesion of myeloma cells to BMSCs was significantly decreased following the inhibition of MUC1 with either siRNA or MUC1 peptide inhibitor, GO201. Treatment of myeloma cells with [Au(d2pype)2]Cl resulted in decreased expression of NF-ĸB and Sp1, which coincided with a decreased expression of MUC1. In conclusion, targeting TrxR with improved gold(I)-based compounds may provide an effective anti-myeloma approach for the future development of MM therapies. Moreover, [Au(d2pype)2]Cl has been shown to be useful for future functional studies that will focus on investigating the mechanisms that are involved in the cellular changes related to the effects from TrxR inhibition.

Multiple myeloma (MM) is the second-most common hematological cancer with poor overall survival in patients. In the past two decades, omics technology has greatly contributed to increased understanding of this disease, including molecular classification, prognostic risk factors, and disease progression. However, despite the tremendous progress, MM is still considered incurable. My work focused on furthering knowledge in this disease by applying computational and statistical methods to investigate the myeloma transcriptome. I used data mining techniques to identify transcription factors associated with myeloma precursor progression and uncovered four transcription factors of interest to be further studied in functional experiments. I also developed a computational means of target selection for precision chimeric antigen receptor (CAR) T-cell therapy in MM patient data and determined translocation subtypes have an influence on target expression. Overall, my work utilized bioinformatics analyses in MM data to identify genes of interest associated with precursor progression and to investigate the suitability of surface targets for CAR T-cell therapy. My research work contributes to the translational work being conducted in MM to determine ways to better understand the disease and improve patient treatment and survival.

Multiple myeloma (MM) is the second most common blood cancer in the United States, having affected approximately 32,000 new patients in 2020.1 It is a plasma cell neoplasm characterized by a pattern of recurrent remissions and relapses. The median 5-year overall survival rate (OS) from diagnosis is only 52%, and ultimately, there is no cure.1 Despite the continual introduction of novel therapies, high dose melphalan and autologous stem cell transplant (HDM-ASCT) remains the standard of care for eligible patients. A challenge with HDM is the wide variability in its exposure and response profiles at the standard dose, likely contributing to suboptimal responses in some, and increased toxicities in others.2 To that end, nearly every patient receiving HDM-ASCT will experience a severe grade of pancytopenia, including neutropenia and thrombocytopenia. While safety is the primary motivation for monitoring blood cell counts post-transplant, there is growing evidence suggesting a possible role for it in describing efficacy as well. The incidence of mild to severe neutropenia has been predictive of improved responses to chemotherapy in several cancers, possibly reflecting a similar cytotoxic effect on the diseased cells.3-5 Similarly, increased melphalan exposure has been associated with greater hematological toxicity and improved progression-free survival (PFS).6,7 On the other hand, a more rapid hematological recovery following HDM-ASCT has also been associated with superior PFS.8-10 Taken together, it may be beneficial to balance the depth and duration of severe cytopenia following HDM-ASCT. A potential strategy to improve the standard HDM-ASCT regimen could involve pharmacokinetic/pharmacodynamic (PK/PD) model-based dose modifications on the basis of predicted cytopenia profiles post-transplant.Population PK/PD modeling and simulation (pharmacometrics) positions itself well with precision medicine, as one of its primary goals is to identify and incorporate various predictive patient factors into the mathematical characterization of drug exposure and response.11,12 An adequate PK/PD model of a drug can predict an individual exposure and response prior to dosing by adjusting the population parameter estimates, determined previously, based on individual covariates, or by updating individual parameters after observing new data as is done in Bayesian forecasting.11 In the case of single HDM-ASCT, a PK/PD model can only make predictions like the former, which heightens the importance of finding the relevant predictors of melphalan exposure and response.In this work, we applied pharmacometrics tools to better understand some of the treatment-related outcomes to HDM-ASCT, specifically post-transplant cytopenias and PFS. We first established best practices in handling missing covariate data by means of a literature review and the results of our simulations in the setting of a population PK analysis. We then developed two models for neutropenia, one more empirically driven and the other more flexible to the initiation of granulocyte-colony stimulating factor (G-CSF) prophylaxis. We then developed a model for thrombocytopenia, and we conclude by proposing these models as potentially useful to improving outcomes to HDM-ASCT.

Multiple Myeloma (MM) is the second most common hematological malignancy, and although patient outcomes have significantly improved since the introduction of autologous stem cell transplantation (ASCT) and novel pharmacological agents such as immunomodulators (IMID), proteasome inhibitors (PI), and monoclonal antibodies (mAb), the disease remains incurable. The pathological complexity of MM results from accumulating mutations in clonal populations of malignant B-cells, which are cytogenetically heterogenous and selectively sensitive to different therapeutic agents. Drug regimens therefore include a diverse combination of therapeutics designed to target specific pathways to inhibit cell proliferation. Recent advances in genomic analytics and novel pharmacological agents potentially allow for more targeted treatments which improve patient outcomes and frequency of remission with minimal adverse effects. Only recently have studies began to correlate an increased understanding of the many subtypes of MM with optimal treatment regimens, and practices such as “Direct to Drug” screening can give clinicians a look at a patient’s likely response to a combination of drugs. By incorporating emerging pharmaceutical agents into studies based on patient characteristics, the management of MM is making incremental strides towards a more targeted treatment paradigm.

BACKGROUND: Multiple myeloma is a rare form of cancer that affects the proper function of plasma cells in the immune system. Patients experience symptoms ranging from bone pain to otherwise avoidable infections that can have negative effects on quality of life. Despite advances in multiple myeloma treatment leading to longer patient survival, it is still an incurable form of blood cancer. As a result, it is important for researchers to constantly investigate new avenues of treatment in order to delay disease progression. This study investigated whether the next generation proteasome inhibitor, ixazomib, could safely delay disease progression in patients who failed a combination regimen that included either the proteasome inhibitor bortezomib or carfilzomib. METHODS: This study is a phase 1/2, 3+3 design, intra-patient, multicenter, open-label, and non-randomized clinical trial that recruited patients that were previously on one of ten combination treatments containing the proteasome inhibitors bortezomib or carfilzomib. Patients must have shown progressive disease while on this treatment in order to qualify. They were given the same drugs and doses they were previously taking except that the proteasome inhibitor was replaced with ixazomib. The safety and efficacy measurements were taken periodically to assess patients’ disease burden. To assess safety, adverse events (AEs) and serious adverse events (SAEs) were recorded, codified, and quantified for analysis. In addition, the maximum tolerated dose (MTS) of ixazomib for three regimens for which it was unknown was investigated through the analysis of dose limiting toxicities (DLTs). Clinical benefit rate (CBR) and overall response rate (ORR) using response data were also determined. Lastly, Kaplan-Meir statistical analysis was used to calculate the secondary efficacy endpoints such as progression free survival (PFS) using data collected throughout the trial. RESULTS: Safety: 24.4% of patients experienced at least one ≥ Grade 3 serious adverse event, 33.3% experienced at least one ≥ Grade 3 adverse event, and two experienced dose limiting toxicities. Efficacy: ORR was 13.2% and the CRR was 18.4%. Median PFS was 2.1 months, duration of response (DOR) was 2.0 months, and overall survival (OS) was 7.9 months. However, the MTD of ixazomib for the three regimens which it was unknown for was not found due to the nature of the data distribution. CONCLUSION: The results indicated that ixazomib is not an effective replacement for bortezomib or carfilzomib in combination treatments containing these drugs, which is apparent from low primary and secondary efficacy endpoints. However, due to a low occurrence of adverse events, serious adverse events, and dose limiting toxicities safety was confirmed. In addition, physicians should determine the MTD on a case by case basis through individual dose escalations if ixazomib is to be used in this context.

Patients with multiple myeloma are generally older and vary in fitness levels, which may influence the clinical benefit of treatment. Patients from the large, phase 3 FIRST trial in newly diagnosed multiple myeloma (NDMM) were retrospectively investigated to determine outcomes based on frailty using scores for age, Charlson Comorbidity Index (CCI), and Eastern Cooperative Oncology Group performance status (ECOG PS), instead of the EQ-5D quality-of-life questionnaire, as previously reported. ECOG PS (n = 1618) was investigated in frailty groups: frail (49%) and nonfrail (51%). Frail patients experienced worse progression-free and overall survival vs nonfrail patients. Prognostic assessment was improved when combining frailty and International Staging System stage (I/II vs III). Frail patients had a higher risk of developing grade 3/4 treatment-emergent adverse events. Treatment effects observed in the FIRST trial were confirmed per frailty group and per frailty and ISS group. The use of this ECOG PS–containing frailty scale as a predictive measure of clinical outcomes in patients with transplant-ineligible NDMM is supported by data from the FIRST trial. This score, based on age, CCI, and ECOG PS, can be easily replicated and may help design future myeloma studies in frail or nonfrail elderly patients.

Daratumumab is a human CD38-targeted monoclonal antibody approved as monotherapy for heavily pretreated relapsed and refractory multiple myeloma. We report findings for the Spanish cohort of an open-label treatment protocol that provided early access to daratumumab monotherapy and collected safety and patient-reported outcomes data for patients with relapsed or refractory multiple myeloma. At 15 centers across Spain, intravenous daratumumab (16mg/kg) was administered to 73 patients who had ≥3 prior lines of therapy, including a proteasome inhibitor and an immunomodulatory drug, or who were double refractory to both. The median duration of daratumumab treatment was 3.3 (range: 0.03–13.17) months, with a median number of 12 (range: 1–25) infusions. Grade 3/4 treatment-emergent adverse events were reported in 74% of patients and included lymphopenia (28.8%), thrombocytopenia (27.4%), neutropenia (21.9%), leukopenia (19.2%), and anemia (15.1%). Common (>5%) serious treatmentemergent adverse events included respiratory tract infection (9.6%), general physical health deterioration (6.8%), and back pain (5.5%). Infusion-related reactions occurred in 45% of patients. The median change from baseline in all domains of the EQ-5D-5L and EORTC QLQ-C30 was mostly 0. A total of 18 (24.7%) patients achieved a partial response or better, with 10 (13.7%) patients achieving a very good partial response or better. Median progression-free survival was 3.98 months. The results of this early access treatment protocol are consistent with previously reported trials of daratumumab monotherapy and confirm its safety and antitumoral efficacy in Spanish patients with heavily treated relapsed or refractory multiple myeloma.

Despite the bone marrow microenvironment being widely recognised as a key player in cancer research, the current animal models that represent a human haematopoietic system lack the contribution of the humanised marrow microenvironment. Here we describe a murine model that relies on the combination of an orthotopic humanised tissue-engineered bone construct (ohTEBC) with patient-specific bone marrow (BM) cells to create a humanised bone marrow (hBM) niche capable of supporting the engraftment of human haematopoietic cells. Results showed that this model supports the engraftment of human CD34+ cells from a healthy BM with human haematopoietic cells migrating into the mouse BM, human BM compartment, spleen and peripheral blood. We compared these results with the engraftment capacity of human CD34+ cells obtained from patients with multiple myeloma (MM). We demonstrated that CD34+ cells derived from a diseased BM had a reduced engraftment potential compared to healthy patients and that a higher cell dose is required to achieve engraftment of human haematopoietic cells in peripheral blood. Finally, we observed that hematopoietic cells obtained from the mobilised peripheral blood of patients yields a higher number of CD34+, overcoming this problem. In conclusion, this humanised mouse model has potential as a unique and patient-specific pre-clinical platform for the study of tumour–microenvironment interactions, including human bone and haematopoietic cells, and could, in the future, serve as a drug testing platform.

11C-methionine (11C-MET) is a new positron emission tomography (PET) tracer for the assessment of disease activity in multiple myeloma (MM) patients, with preliminary data suggesting higher sensitivity and specificity than 18F-fluorodeoxyglucose (18F-FDG). However, the value of tumor burden biomarkers has yet to be investigated. Our goals were to corroborate the superiority of 11C-MET for MM staging and to compare its suitability for the assessment of metabolic tumor burden biomarkers in comparison to 18F-FDG. Twenty-two patients with newly diagnosed, treatment-naïve symptomatic MM who had undergone 11C-MET and 18F-FDG PET/CT were evaluated. Standardized uptake values (SUV) were determined and compared with total metabolic tumor volume (TMTV) for both tracers: total lesion glycolysis (TLG) and total lesion 11C-MET uptake (TLMU). PET-derived values were compared to Revised International Staging System (R-ISS), cytogenetic, and serologic MM markers such as M component, beta 2 microglobulin (B2M), serum free light chains (FLC), albumin, and lactate dehydrogenase (LDH). In 11 patients (50%), 11C-MET detected more focal lesions (FL) than FDG (p < 0.01). SUVmax, SUVmean, SUVpeak, TMTV, and TLMU were also significantly higher in 11C-MET than in 18F-FDG (p < 0.05, respectively). 11C-MET PET biomarkers had a better correlation with tumor burden (bone marrow plasma cell infiltration, M component; p < 0.05 versus p = n.s. respectively). This pilot study suggests that 11C-MET PET/CT is a more sensitive marker for the assessment of myeloma tumor burden than 18F-FDG. Its implications for prognosis evaluation need further investigation.

Multiple myeloma (MM), also known as plasma cell myeloma, is a hematological cancer. In 2003, the first proteasome inhibitor BTZ was approved by the U.S. Food and Drug Administration to treat MM. This dipeptide boronic acid analogue bortezomib (BTZ) is a proteasome inhibitor that targets the 26S proteasome complex and became an effective anti-MM drug. However, despite this encouraging progress, drug resistance remains a prominent challenge to myeloma therapy using BTZ. MM is still considered to be an incurable disease. Therefore, it is important to understand the mechanism of action of BTZ and the mechanism of BTZ resistance in MM. The essence of the mechanism of the action of BTZ is the inhibition of the proteasome, which inhibits the protein degradation of the ubiquitin-proteasome pathway. This inhibition results in the blocking of the degradation of abnormal proteins, thus triggering ER stress and apoptosis and the blocking of the degradation of key regulatory proteins, thus directly altering pro-survival or pro-apoptotic signalling cascades. The mechanisms of BTZ resistance is complicated, which include genetic abnormalities in the proteasome, use of an alternative protein degradation pathway, aberrant survival signalling pathways, metabolism, microenvironment, and drug efflux such as via P-glycoprotein (Pgp). The role of Pgp in BTZ resistant MM has been studied. It has been shown that BTZ is one of the substrates of Pgp. However, the correlation between Pgp and BTZ resistance in MM conflicts in different studies. This study found that the mRNA level and activity level of Pgp were upregulated, and the inhibition of Ppg by tariquidar overcame the BTZ resistance in both acquired and hypoxia-induced RPMI8226 and U266 myeloma cells. These results suggest that the Pgp indeed plays a role in the BTZ resistance in MM. Recent studies showed that carbonic anhydrase XII (CAXII) is overexpressed in the Pgp-positive chemoresistant lung, osteosarcoma, and colon cancer cells and its activity is necessary for the activity of Pgp, and the inhibition of CAXII overcomes the Pgp-mediated chemoresistance. This study showed that The CAXII inhibitor Psammaplin C reduced the Pgp activity and overcame the BTZ resistance in the acquired and hypoxia-induced RPMI8226 and U266 myeloma cells. These results support that CAXII plays a role in the BTZ resistance in MM. Previous structural studies showed that both Pgp and CAXII contain a disulfide bond, which allows the possibility that they may be regulated by the thioredoxin (Trx) system. This study showed that the inhibition of Trx system by auranofin reduced the Pgp expression levels in the hypoxia-induced BTZ resistant RMI8226 and MM cell and also reduced the mRNA levels of CAXII in the hypoxia-induced BTZ resistant RPMI8226 and U266 myeloma cells. These results support that the Trx system could be capable of regulating the expression of Pgp and CAXII in MM. Based on the results obtained with Pgp and CAXII, the expression levels of Pgp and CAXII in MM patients were also analysed using bioinformatics methods. Using publically available datasets this study showed Pgp and CAXII have higher expression in MM patients who respond to BTZ than the patients who do not respond to BTZ. These results support that CAXII and Pgp play an important role in BTZ resistance in MM. In addition, bioinformatics analysis highlighted 327 differently expressed genes in the relapsed MM patients. The interactions analysis by the Ingenuity Pathway Analysis program highlighted potential targets for BTZ resistance in the relapsed MM cells including NFκB, ElK1, HOXB9, CALM, and Fyn. In conclusion, this project highlights the role of Pgp and CAXII in the resistant MM cells. This project also showed that the Trx system could be capable of regulating the expression of Pgp and CAXII in MM. Bioinformatics analysis highlights 327 differently expressed genes in the relapsed MM patients compared to newly diagnosed MM patients and potential targets for BTZ resistance including nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB), ETS Like-1 protein (ElK1), Homeobox B9 (HOXB9), calmodulin (CALM), and tyrosine-protein kinase (Fyn).

The proteasome is central to cellular protein regulation by degrading proteins conjugated to multiple units of the polypeptide ubiquitin. Due to their involvement in the regulation of apoptosis in cells and their abundance in multiple myeloma cells (MM), the proteasome has become a target for cancer treatment. Aza-peptide inhibitors are a new class of inhibitors designed to inhibit the proteasome and other proteases where a P1-amino acid and an electrophilic warhead comprise the potential therapeutic. Previous inhibitors of this type exhibited competitive inhibition of the 20S proteasome with Ki values in the micromolar range for in vitro studies and an IC50 value of 10–50 µM when screened against multiple myeloma and natural killer cell lines.49 In this thesis we present the synthesis and initial SAR studies of different sequences of proteasome-specific aza-peptide inhibitors with ketones and Michael acceptor warheads. We also present the tunability of these inhibitors by designing and synthesizing caspase-6 specific inhibitors. Our goals are to improve the potency and selectivity of aza-peptide inhibitors for optimal selectivity to their target enzymes.

Abstract: Multiple myeloma (MM) is a hematological malignancy which is caused by aberrant accumulation of monoclonal plasma cells (PCs) within the bone marrow. The oncogenesis of MM is mediated by chromosomal aberrations as well as the tumor microenvironment in the bone marrow. Signal transducer and activator of transcription 3 (STAT3) is active in more than 50% of MM patients, and it is known to induce cell growth, survival and drug resistance in MM cells. However, activation of STAT3 in MM cells is largely dependent on exogenous soluble factors and cell adhesion. Conventional culture method is therefore not an ideal model to recapitulate the in vivo STAT3 activity of MM cells. In this thesis, I explored the importance of STAT3 in MM cells with the consideration of MM tumor microenvironment using a 3D culture model. Moreover, I aimed to develop a method for better delivery and efficacy of STAT3 inhibitor in vivo as a therapy for MM. I hypothesize that STAT3 activity in MM cells is more pronounced in close-to-in vivo tumor microenvironment, hence improvement of STAT3 inhibitor delivery by nanoparticle conjugation and anti-CD38 conjugation is a valid therapy for MM. A 3D scaffold culture model for MM cells was established for long-term culture of primary MM cells by Kirshner et al. However, the biochemical and biological effects on MM cells in this 3D model is not known. Two MM cell lines were found to have a higher level of active STAT3 (by means of phosphorylated STAT3 or pSTAT3) in the 3D culture model were compared with their counterparts in conventional culture. This elevated pSTAT3 level was dependent on the 3D environment since it decreased soon after transferring to conventional culture. STAT3 inhibition using a pharmacological agent, Stattic, significantly decreased the cell viability of MM cells and sensitized them to bortezomib in 3D culture but not in conventional culture. Using an oligonucleotide array, 3D cultured MM cells showed increased expression of several known STAT3 downstream genes implicated in oncogenesis. Primary multiple myeloma (PMM) cells harvested from patients are highly valuable resources for studying the biology of the disease and drug resistance. However, the use of these cells is limited by the fact that PMM cells are short-lived in conventional culture. The role of STAT3 and 3D culture system on prolonging the longevity of PMM cells was evaluated. Using PMM cells from patients, a significantly higher total viable PMM cell number in 3D compared to conventional culture was observed. However, the MM cell proliferation rate in the two culture systems was similar. Correlating with the high MM cell viability, higher pSTAT3 level was also observed in 3D culture. After treatment of IL6, the PMM cell viability in 3D culture was further improved. Treatment of Stattic on 3D cultured PMM cells significantly ablated their viability. On the other hand, treatment of IL6 or Stattic did not change the cell viability of conventionally cultured PMM cells. Many STAT3 inhibitors have been developed, but none of them has been approved as a cancer therapy due to their hydrophobicity and severe off-target toxicity. Lavasanifar Lab has developed a nanoparticular formation of a STAT3 inhibitor, S3I-1757 (Null-S3I-NP) for better delivery and in vivo efficacy. This nanoparticular formulation was conjugated with CD38 monoclonal antibody (denoted as CD38-S3I-NP) to further increase its MM cell targeting ability and anti-MM efficacy. CD38-S3I-NP is slightly less stable than Null-NP with a higher S3I-1757 release rate within a short time. However, CD38-conjuagred nanoparticles showed significantly higher MM cellular uptake than compared to plain nanoparticles. In keeping with this, CD38-S3I-NP resulted in a significantly lower IC50 value in two IL6-stimulated MM cell lines compared to Null-S3I-NP. CD38-S3I-NP suppressed MM tumor growth more effectively compared to Null-S3I-NP in vivo. The pSTAT3 level in the bone marrow mononuclear cells was significantly reduced in MM-bearing mice after CD38-S3I-NP treatment compared to Null-S3I-NP. These findings suggest that CD38-S3I-NP is potent anti-STAT3 agent with MM cell-targeting ability. In conclusion, this thesis reveals the essential role of STAT3 activity in maintaining the viability of MM cells in the context of 3D microenvironment. Therefore, MM cell-targeting nanoparticles with STAT3 inhibitor is believed to be a promising therapy for MM.

Oncology clinical trials generate the evidence required to obtain regulatory approval for new interventions; the life-saving treatments cancer patients receive today, and the novel therapies that will transform future care paths, rely on data from clinical trials. Unfortunately, the therapeutic advances driven by clinical research are limited to the patient populations that best represent those enrolled in clinical trials. The explicit driver of this limited generalizability is the design of clinical trial eligibility criteria. Although overly restrictive eligibility criteria have been critiqued in the literature, quantitative studies evaluating the appropriateness of these criteria have not been performed. Therefore, we analyzed the eligibility criteria of a particular oncology disease group (hematologic malignancies), specifically exploring: 1) the relationship between commonly used organ function eligibility criteria and the expected toxicities of the trials’ interventions, 2) reasons for ineligibility and the outcomes of leukemia patients eligible vs. ineligible for South Western Oncology Group (SWOG) trials, and 3) the health policy implications of overly restrictive eligibility criteria. Collectively, the findings of these studies suggest that the eligibility criteria for hematologic malignancy clinical trials are overly restrictive; the organ function criteria fail to reflect the expected toxicities of the trials’ interventions / observed adverse events and the administrative criteria associated with the timing of screening tests / sample submissions included in SWOG leukemia protocols are too conservative. Furthermore, our results demonstrated the safety and efficacy outcomes were comparable between the leukemia/myelodysplastic syndrome patients ineligible for administrative or non-clinically significant reasons and the patients fully meeting the eligibility criteria. These findings suggest, patients who may benefit from potentially life-saving treatments available through clinical trials are denied access to these interventions. Consequently, overly restrictive eligibility criteria, resulting in the systemic exclusion of specific patient populations, diminishes the generalizability of the trials’ results, leading to poorly designed health policy decisions, particularly within the context of value-based provider reimbursement.

Daratumumab is an anti-CD38 fully human IgG1 mAb approved for multiple myeloma treatment. One of the proposed mechanisms of action is the induction of antibody-dependent cellular cytotoxicity (ADCC) mediated by NK cells. NK cells acquire surface CD137 expression in the presence of solid-phase-attached daratumumab and when encountering a daratumumab-coated CD38+ tumor cell line. In this setting, addition of the agonist anti-CD137 mAb urelumab enhances NK-cell activation increasing CD25 expression and IFNɣ production. However, in vitro ADCC is not increased by the addition of urelumab both in 4h or 24h lasting experiments. To study urelumab-increased daratumumab-mediated ADCC activity in vivo, we set up a mouse model based on the intravenous administration of a luciferase-transfected multiple myeloma cell line of human origin, human NK cells and daratumumab to immuno-deficient NSG mice. In this model, intravenous administration of urelumab 24h after daratumumab delayed tumor growth and prolonged mice survival.

Multiple myeloma (MM) is a plasma cell dyscrasia characterized by bone marrow infiltration of clonal plasma cells. The recent literature has clearly demonstrated clonal heterogeneity in terms of both the genomic and transcriptomic signature of the tumor. Of note, novel studies have also highlighted the importance of the functional cross-talk between the tumor clone and the surrounding bone marrow milieu, as a relevant player of MM pathogenesis. These findings have certainly enhanced our understanding of the underlying mechanisms supporting MM pathogenesis and disease progression. Within the specific field of small non-coding RNA-research, recent studies have provided evidence for considering microRNAs as a crucial regulator of MM biology and, in this context, circulating microRNAs have been shown to potentially contribute to prognostic stratification of MM patients. The present review will summarize the most recent studies within the specific topic of microRNAs and circulating microRNAs in MM.

Multiple myeloma (MM) remains as an incurable disease and, although allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potentially curative approach, most patients ultimately relapse, and their treatment remains challenging. Because allo-HSCT can modify not only the biology of the disease, but also the immune system and the microenvironment, it can potentially enhance the response to rescue therapies. Information on the efficacy and safety of novel drugs in patients relapsing after allo-HSCT is lacking, however. The objectives of this study were to evaluate the efficacy and toxicity of rescue therapies in patients with MM who relapsed after allo-HSCT, as well as to compare their efficacy before and after allo-HSCT. This retrospective multicenter study included 126 consecutive patients with MM who underwent allo-HSCT between 2000 and 2013 at 8 Spanish centers. All patients engrafted. The incidence of grade II-IV acute graft-versus-host disease (GVHD) was 47%, and nonrelapse mortality within the first 100 days post-transplantation was 13%. After a median follow-up of 92 months, overall survival (OS) was 51% at 2 years and 43% at 5 years. The median progression-free survival after allo-HSCT was 7 months, whereas the median OS after relapse was 33 months. Patients relapsing in the first 6 months after transplantation had a dismal prognosis compared with those who relapsed later (median OS, 11 months versus 120 months; P < .001). The absence of chronic GVHD was associated with reduced OS after relapse (hazard ratio, 3.44; P < .001). Most patients responded to rescue therapies, including proteasome inhibitors (PIs; 62%) and immunomodulatory drugs (IMiDs; 77%), with a good toxicity profile. An in-depth evaluation, including the type and intensity of PI- and IMiD-based combinations used before and after allo-HSCT, showed that the overall response rate and duration of response after allo-HSCT were similar to those seen in the pretransplantation period. Patients with MM who relapse after allo-HSCT should be considered candidates for therapy with new drugs, which can achieve similar response rates with similar durability as seen in the pretransplantation period. This pattern does not follow the usual course of the disease outside the transplantation setting, where response rates and time to progression decreases with each consecutive line of treatment.

Multiple myeloma is a hematological malignancy characterized by clonal proliferation of plasma cells generally caused by chromosomal abnormalities. It occurs in the bone marrow, which is the microenvironment of multiple myeloma. Exosomes (EXs) are 30-100 nm membrane-derived micro-vesicles containing various of bioactive molecules, such as microRNAs, to mediate the cell-cell interaction. Numerous studies reported that exosomes play a significant role in tumor microenvironment. Angiogenesis has the important implication in tumor exacerbation to supply nutrients to promote the progression of cancer cells through endothelial cells (ECs). Some studies demonstrated that microRNA-29b (miR-29b) can suppress tumor development and inhibit angiogenesis. Therefore, in this study, we designed experiments to research the relationship between exosomes released from multiple myeloma, miR-29b and angiogenesis in ECs. Two types of multiple myeloma cells, OPM2 and RPMI-8226 cell lines, were treated with C6-Ceramide. Their released exosomes (MM-EXC6-Cer) were collected, which enriched in miR-29b. MM-EXC6-Cer were cocultured with human umbilical vein endothelial cells (HUVECs) to test the effect on angiogenic function of HUVEC. The results showed that the EC proliferation, the tube formation, migration and vascular endothelial growth factor A (VEGFA) expression were decreased in ECs. In addition, miR-29b inhibitor was used in ECs, and could decrease the level of the miR-29b in ECs. Exosomes released from multiple myeloma cells (MM-EX) were cocultured with ECs, which were treated with miR-29b inhibitor, to examine the effects on EC angiogenic function. We found that EC proliferation, VEGFA expression, migration and tube formation were promoted. This data demonstrated that miR-29b can negatively modulate the angiogenic function of ECs through exosomes secreted by multiple myeloma cells.

Multiple myeloma is a neoplasm of plasma cells which develops in the skeleton. The bone microenvironment supports myeloma growth and long-term survival of dormant myeloma cells. Despite targeted treatments, patients relapse and survival is poor. Dormant cells contribute to disease relapse and represent potential therapeutic targets. However, the molecular pathways that control dormancy are unclear. We developed a method to identify dormant cells in vivo and have used both microarray and single cell RNA sequencing (scRNA-seq) approaches to define the transcriptome profile of these cells. To identify dormant myeloma cells in the skeleton, 5TGM1 murine myeloma cells expressing eGFP were labelled with a fluorescent membrane dye, (Vybrant DiD). Dividing cells share DiD with daughter cells which DiD is diluted and activated cells become DiDneg, whereas, non-dividing dormant cells retain DiD (DiDhigh). DiD-labelled cells were injected (i.v.) into C57BLKalwRij mice. After 21 days, individual DiDhigh and DiDneg cells were sorted into 384 well plates by FACS for scRNA-seq and sequenced at >2x10^6 reads/cell. 5TGM1 cells were validated by the reconstruction of the unique clonal idiotypic sequence and the expression of specific Ig genes. A mean of ~4000 unique transcripts were identified with over 900 genes up-regulated in DiDhigh cells compared to DiDneg cells (fold change >2). Functional annotation of differentially expressed genes confirmed the dormant status of DiDhigh cells with down-regulation of metabolic pathways and up-regulation of immune system-related pathways. We identified the receptor tyrosine kinase, Axl, as a potential therapeutic target. FACS analysis confirmed expression in dormant cells and analysis of CD138+/CD38+ plasma cells from myeloma patients identified a small population of cells express AXL. Inhibition of Axl with Cabozantinib or BMS-777607 decreased dormant cell numbers and increased tumour burden in myeloma bearing mice suggesting Axl is functionally important in dormancy retention. Moreover, survival analysis in independent patient cohorts correlated the dormancy signature expressed in dormant myeloma cells with improved survival. These data demonstrate that dormant myeloma cells persist in the skeleton, have a unique transcript profile and this knowledge can be used to predict survival and identify new targets that alter the behaviour of myeloma disease in the skeleton.