Common Hematologic Malignancy Research Articles

Multiple myeloma and the potential of new checkpoint inhibitors for immunotherapy

Multiple myeloma (MM), a cancer of the bone marrow, is categorized as the second most common hematological malignancy of adults in the Western world. Despite dramatic improvements in immunotherapies in the field of cancers, MM immunotherapy has not been promising until now. Recent clinical studies of immune checkpoint inhibitor therapy, either alone or in combination with anticancer drugs, showed excessive side effects or low efficacy, particularly in advanced MM patients. In this context, lymphocyte levels of exhaustion markers play a pivotal role in the MM tumor microenvironment (TME). Hence in the present review, the mechanisms relevant to MM of five inhibitory molecules including T-cell immunoreceptor with Ig and ITIM domains (TIGIT), T-cell immunoglobulin, and mucin domain 3 (Tim-3), lymphocyte activation gene-3 (LAG-3), V-domain Ig Suppressor of T-cell activation and killer immunoglobulin-like receptors along with bispecific T-cell antibodies (BsAbs) will be discussed. Further, we summarized the underlying biology of these checkpoints in cancer and their rapidly emerging role in pathways in MM along with presenting recent clinical trials in context.

Genomic Analysis of Lymphoma Risk in Bullmastiff Dogs.

Lymphoma is the most common haematological malignancy affecting dogs and has a high incidence in the Bullmastiff breed. The aim of this study was to identify risk loci predisposing this breed to the disease. The average age of lymphoma diagnosis in 55 cases was less than 6 years, similar to the median age of 64 cases from our clinical and pathology databases. When fine-scale population structure was explored using NETVIEW, cases were distributed throughout an extended pedigree. When genotyped cases (n = 49) and dogs from the control group (n = 281) were compared in a genome-wide association analysis of lymphoma risk, the most prominent associated regions were detected on CFA13 and CFA33. The top SNPs in a 5.4 Mb region on CFA13 were significant at a chromosome-wide level, and the region was fine-mapped to ~1.2 Mb (CFA13: 25.2-26.4 Mb; CanFam3.1) with four potential functional candidates, including the MYC proto-oncogene bHLH transcription factor (MYC) and a region syntenic with the human and mouse lncRNA Pvt1 oncogene (PVT1). A 380 Kb associated region at CFA33: 7.7-8.1 Mb contained the coding sequence for SUMO specific peptidase7 (SENP7) and NFK inhibitor zeta (NFKBIZ) genes. These genes have annotations related to cancer, amongst others, and both have functional links to MYC regulation. Genomic signatures identified in lymphoma cases suggest that increased risk contributed by the regions identified by GWAS may complement a complex predisposing genetic background.

Exosomal miR-182 derived from bone marrow mesenchymal stem cells drives carfilzomib resistance of multiple myeloma cells by targeting SOX6

BackgroundMultiple myeloma (MM) is a common hematological malignancy. Drug resistance remains to be a major clinical challenge in MM therapy. In this study, we aim to investigate the functional roles of bone marrow mesenchymal stem cells (BMSC)-derived exosomal miR-182 on the carfilzomib resistance of MM and its underlying mechanism.MethodsqRT-PCR and Western blot methods were utilized to confirm the gene or protein expressions. CCK-8 and transwell assays were performed to measure the capabilities of proliferation, migration, and invasion. The molecular interactions were validated through ChIP and Dual luciferase assay.ResultsOur findings indicated that miR-182 expression was upregulated in serum, BMSCs and BMSC-derived exosomes from MM patients. Hypoxia-inducible factor-1α (HIF-1α), a key transcriptional factor in tumor microenvironment, could boost miR-182 expression by directly binding to its promoter, thus favoring exosomal secretion. Moreover, exosomal miR-182 from BMSCs could be transferred to MM cells and was able to promote malignant proliferation, metastasis, and invasion, as well as decrease the sensitivity of MM cells against carfilzomib. Additionally, SOX6 was identified as a downstream target of miR-182 in MM cells, and its expression was negatively regulated by miR-182. Rescue experiments proved that loss of SOX6 in MM cells dramatically reversed the promoting roles of BMSC-secreted exosomal miR-182 on proliferation, metastasis, and carfilzomib resistance in MM cells.ConclusionCollectively, our findings indicated that exosomal miR-182 derived from BMSCs contributed to the metastasis and carfilzomib resistance of MM cells by targeting SOX6. This study sheds light on the pathogenesis of the BMSC-derived exosome containing miR-182 in the malignant behaviors of MM cells and carfzomib resistance.

Abstract C084: Metabolomic profiling identifies molecular modifiers contributing to proteasome inhibitor resistance in multiple myeloma

Abstract Metabolic reprogramming plays a crucial role in the maintenance and differentiation of various cancers, including multiple myeloma (MM), the second-most common incurable hematological malignancy. Drug resistance poses a significant challenge to conventional proteasome inhibitor (PI) based therapy. A body of literature suggests that several metabolites are significantly deregulated in MM patients, however, their role in driving resistance is unclear. This study aims to identify metabolites modulated during acquired resistance to proteasome inhibitors in relapsed refractory (RR)MM. Two clinically relevant cell line models of PI-resistance (PIR) were developed by dose-escalation of the PI over one year. Cell cytotoxicity assays, immunoblotting, immunofluorescence, and flow cytometric analyses were used to assess the resistance phenotype. Mass spectrometry-based metabolomic profiling was performed to identify altered metabolites in resistant as compared to sensitive cell lines. PIR cells demonstrated pan-resistance to multiple PIs, including a >10-fold increase in IC50 for oprozomib compared to parental cells. PIR cells had higher bioenergetic and glycolytic activity (Seahorse) while gene expression profiling suggested downregulation of oxidative phosphorylation. Mass spectrometry-based metabolomic profiling identified distinct metabolites deregulated in drug-resistant cells. The metabolomic profiles of the drug-resistant cell lines showed that the lipid synthesis and beta-oxidation of fatty acid pathways were highly modulated. Several pathway components demonstrated a negative association with overall survival in multiple myeloma patient datasets (MMRF, GSE31161 & GSE9782). PI resistance led to increased phosphorylation of ACLY involved in fatty acid synthesis and increased expression of CPT1A indicative of activation of the beta-oxidation pathway. Use of ACLY and FASN tool compounds affected proliferation, survival, and response to treatment in PIR cells. Metabolites modulated during acquired resistance to proteasome inhibitor in relapsed refractory (RR)MM were identified and potential mechanisms driving resistance were studied. Citation Format: Snehal M Gaikwad, Vincent Keith Hughitt, Nana Gyabaah-Kessie, Mikaela Matera-Vatnick, Aleksandra A Michalowski, Anaisa Quintanilla-Arteaga, Thorkell Andresson, Beverly A Mock. Metabolomic profiling identifies molecular modifiers contributing to proteasome inhibitor resistance in multiple myeloma [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C084.

Demographic Characteristics and Treatment Patterns in Veterans with Multiple Myeloma: A Comprehensive Analysis from the Veterans Affairs Database (2000-2020)

BACKGROUND: Multiple Myeloma (MM) is the 2nd most common hematologic malignancy, accounting for 32,270 new cases diagnosed in the United States each year with over 12,830 deaths and a 57% overall 5 -year survival rate. Increased incidence is seen in patients aged >60, African American (AA) race and Male population. Despite significant advancements in therapies with novel agents and immunotherapy, MM remains a challenging disease to manage due to its heterogeneity, resistance to treatment, and the risk of relapse. In this study, we aim to determine the demographics, comorbidities and trends in treatment patterns of MM patients, over time in the VA database. METHODS: Patients diagnosed with MM from 2000-2020 in the VA's nationwide electronic health records were identified using the VA Corporate Data Warehouse (VA CDW) and VINCI to host and analyze the data. Patients were identified using International Classification of Diseases codes for MM. Patient demographics, including drugs and therapies used, were identified through structured data in the VA CDW. We included all veterans 1) over the age of 18, 2) diagnosed with MM with 3 coded visits on different days and received 1 agent other than steroids after diagnosis date. We excluded all patients who did not have at least two VA primary care visits, or one primary care visit and one VA pharmacy refill in the one year prior to MM diagnosis. We analyzed demographic details, comorbidity burden, treatment regimens for induction and attrition rates. RESULTS: A total of 12,196 patients were identified, with a median age at diagnosis of 68 years (range, 28-96 years), of which 97.8% were males, 59% (n=7187) were White, 23% (n=2903) were Black, Other (Asian/Pacific Islanders) made up 1.9% the cohort (n=233) and for 15% cohort (n=1867), race was either unknown or missing. Comorbidities at diagnosis were the following: 4164 patients had diabetes mellitus; 1329 patients had CHF ,2159 patients had pre-existing renal disease. 10,156 patients received at least one line of therapy, with the following distribution: 215 pts - RVD, 20 - DRD, 344 - CyBORD , 1930 - oral Melphalan, 3368- Thalidomide, 2692 -Revlimid/Dexamethasone, 1555 - Velcade/Dexamethasone and 2040 patients received an induction regimen not specified in Table 2. The breakdown of first line therapy utilization in respect to race is shown in Table 2. 4228 patients received two distinct lines of therapy (LOT), 1718 patients received 3 distinct LOT and 419 patients received 4 LOT. Transitioning from 1st st to 2nd LOT, attrition rates of approximately 50.3% for Black and 56.5% for White patients were observed, and from two to three lines, rates were around 52.4% for Black and 57.8% for White patients, indicating substantial drop-offs in treatment progression. The Cox proportional hazard model analysis reveals significant predictors for overall survival. Notably, age (70 or older) and renal disease was associated with increased mortality with hazard ratios (HR) of 1.731 and 1.275 (p < .0001 for both), respectively. Conversely, Black and White races, and receiving three or more therapy lines, was associated with increased OS, with HRs of 0.545, 0.607, and 0.825 (p < .0001 for all). CONCLUSIONS: This comprehensive overview of veterans with MM in the VA system over a 20-year period reveals heterogeneity in treatment utilization as expected with the changing landscape of MM treatment. A detailed analysis on the nature of subsequent lines of therapies, including stem cell transplant utilization, access to clinical trials is ongoing and will be submitted as a full manuscript publication. Additionally, the data on the staging and cytogenetics is also being obtained.

E3 Ubiquitin Ligase TRIM21 Enhances Macrophage-Mediated Bortezomib Resistance By Inducing M2 Polarization in Multiple Myeloma

Background: Multiple myeloma (MM) is a malignant plasma cell hematologic tumor with an increasing incidence and has become the second most common hematologic malignancy after non-Hodgkin lymphoma. Macrophage is an abundant component of the MM bone marrow microenvironment and contributes to the enhancement of MM drug resistance. Our previous study found that the expression of the E3 ubiquitin ligase TRIM21 was reduced in MM cells, and MM cells with low expression of TRIM21 showed reduced sensitivity to Bortezomib (Bort). This study aims to investigate the role and mechanism of TRIM21 in macrophage-mediated MM Bort resistance. Objective ：Explore the effect and mechanism of TRIM21 in mediating multiple myeloma Bortezomib resistance. Methods ：1. Immunofluorescence to analyze the expression of TRIM21 on macrophages in bone marrow biopsy specimens from patients with MM; 2. QPCR, western blot (WB) and flow cytometry (FCM) to detect the level of macrophage polarization markers after constructing overexpression or knockdown of TRIM21; 3. WB and FCM to detect the sensitivity of MM cells to Bort after co-culture of MM cells and macrophage; 4. Transcriptome sequencing to identify TRIM21-mediated pathway changes in macrophage; 5. QPCR and WB to verify the effect of TRIM21 on macrophage polarization after treatment with pathway inhibitors. Results:1. Immunofluorescence analysis showed increased expression of TRIM21 in macrophage from relapsed and refractory MM (RRMM) patients compared to newly diagnosed multiple myeloma (NDMM) patients;2. QPCR, WB and FCM results showed that overexpression of TRIM21 in macrophage led to decreased polarization markers of M1 phenotype and increased the polarization markers of M2 phenotype. In contrast, knockdown of TRIM21 increased M1 polarization and decreased M2 polarization; 3. WB and FCM results demonstrated that co-culture of MM cells with TRIM21-overexpressing macrophages significantly enhanced MM cell tolerance to Bortezomib and decreased MM cell apoptosis; 4. Transcriptome sequencing revealed activation of the JAK-STAT3 pathway in macrophages after TRIM21 overexpression; 5. QPCR and WB results showed that the macrophage polarization effect induced by TRIM21 overexpression was reversed after treatment with the STAT3 inhibitor C188-9. Conclusions: Our study shows that TRIM21 is overexpressed in macrophages in patients with relapsed and refractory MM. In addition, TRIM21 induces M2 polarization by activating the JAK-STAT3 pathway, thereby enhancing the protective effect of macrophage on MM cells. These findings provide a new insight into the role of TRIM21 in MM drug resistance and lay an important theoretical foundation for targeting macrophages in MM. Key words: TRIM21, Macrophage, Bortezomib, JAK-STAT3

Artificial Intelligence of the 2-D and 3-D Bone Marrow Microenvironment to Identify Cytogenetic Subtypes of Multiple Myeloma

Multiple myeloma is the second most common hematologic malignancy with an annual incidence of 35,000 new cases every year in the U.S. Due to improved access to effective therapies, overall survival has improved from 1-3 years to over 10-20 years. However, there is not yet any available curative therapy for multiple myeloma and most patients will suffer from recurrent relapses, with an unmet clinical need for novel therapies. The interplay between the bone marrow microenvironment and malignant plasma cells is critically important to better understand pathogenesis of the disease, to predict response to therapy (in particular, immunotherapy), and to identify novel drug targets. Leveraging the power of computer vision, we are developing novel machine learning algorithms to label and characterize individual cells from digitalized H&E and CD138 stained plasma cells in bone marrow core biopsies. A total of 188 patients were included in this analysis: 157 with multiple myeloma, 6 with monoclonal gammopathy of undetermined significance, 17 with smoldering multiple myeloma, and 6 with primary amyloidosis. The deep learning framework to build our artificial intelligence (AI) models is implemented by Pytorch. Using the surface creation tool from the Imaris 10.0.1 software, we are able to measure pixel intensity, area, sphericity, and other cellular features that are then correlated with myeloma clinical outcomes as well as defined cytogenetic subtypes of multiple myeloma in 2-D. As a preliminary step in this project we analyzed the IMARIS software's ability to accurately determine the number of plasma cells compared with clinical standards through a linear regression plot with strong correlation (R 2=0.9). In addition, we rendered the 3-D microenvironment by combining more than 60 sequential formalin-fixed paraffin embedded (FFPE) tissue block bone marrow biopsy samples sliced at a thickness of 4 microns. Through utilizing the surface creation tool on the 2-D images within the 3-D model, we can show distance measurements between plasma cells and other features of the microenvironment such as adipocytes. This insight allows us to better understand and control the immune microenvironment through observing the various 2-D tissue slices within the 3-D model. Furthermore, we have assessed whether multiple myeloma digital images are able to detect translocation [t(11;14)] multiple myeloma subtype, which is present in 15% to 24% of patients, shown to predict treatment response to Bcl-2 inhibitor therapy (such as venetoclax). A small cohort (n=25) of patients with t(11;14) have been compared to non-t(11;14) cases using the machine learning model and preliminary data indicate that there is a correlation between digital images and multiple myeloma subtype. In the future, we anticipate our AI model will improve the accuracy of diagnostic methods while reducing costs and offering deeper translational insights that will predict drug response in vivo.

Therapy Related Multiple Myeloma: A Distinct Entity

Background The development of therapy-related multiple myeloma (t-MM) is a poorly described entity. Multiple myeloma (MM) is the second most common hematologic malignancy. Despite significant strides in MM treatment over recent decades, identifying modifiable risk factors, optimizing screening methodologies, and identifying potential treatment strategies or targets to address t-MM remains of utmost importance, as this disease continues to pose a significant therapeutic challenge and remains incurable. This retrospective review seeks to explore the incidence and characteristics of t-MM at a medium-sized, academic center. Methods We performed a single institution retrospective chart review of patients diagnosed with multiple myeloma between 2008-2023 and evaluated for prior diagnosis of solid malignancy, excluding all skin cancers, lymphomas or myeloproliferative neoplasm (MPN). We collected data for the following: prior treatment for solid malignancy, lymphoma or MPN, lapsed time between prior therapy and development of MM, cytogenetics, prominent subtype present, renal failure and hypercalcemia if present at time of diagnosis, management of t-MM including transplant data, disease progression including relapse status, and survival data. Results In our study, we identified a subgroup of 17 individuals who had a previous history of solid malignancy, 1 patient with unknown prior solid tumor, 1 patient with history of Hodgkin's lymphoma, and 1 additional patient with history of MPN who received chemotherapy and/or radiation therapy, with 3 patients having MGUS prior to development of MM. Notably, among this population, 70% were male. All patients developed MM following the treatment for a solid malignancy, lymphoma or MPN. The average amount of time from diagnosis of solid tumor, lymphoma or MPN to development of MM was 9.53 years (1-25 years). 50% of the individuals were White Americans, while the remaining 50% were African Americans. A significant majority (85%) of the population had a single primary solid tumor. Among the primary solid tumors, 45% were prostate cancer, 15% were breast cancer, and 10% were renal cell carcinoma (Figure 1). 5 patients received chemotherapy alone without documented prior radiation exposure while 13 had prior radiation exposure for prior solid malignancy. At time of diagnosis, 45% of patients had renal failure with 10% of patients having both renal failure and hypercalcemia. Within the t-MM subtypes, we discovered that 50% had IgA type, with 70% of these individuals exhibiting the IgA Kappa subtype. 45% of the individuals who developed t-MM had the IgG type, with 78% of them with IgG Kappa subtype. 15 (75%) of the patients received transplant and only 2 patients had confirmed relapse with 2 patients unknown and 2 additional patients who passed away prior to starting maintenance therapy following transplant. 4 patients are very early in their disease process and as of yet have not undergo transplant. 55% of our patients are presumed living. The OS for patients with t-MM was 1190 days compared to the average OS of 2326 days in our institutional MM database without t-MM. Conclusion Our analysis focused on patients diagnosed with MM who had a prior history of solid primary tumors, lymphoma or MPN treated with chemotherapy and/or radiotherapy in hopes of providing valuable insights into the characteristics and outcomes of individuals who developed MM following treatment for solid tumors, lymphoma or MPN. Our findings highlighted specific risk factors associated with t-MM in this population. Being male and having a history of prostate cancer was associated with higher incidence of development of t-MM. This is likely related to the radiation therapy associated with prostate cancer, as radiation exposure has been a known risk factor for the development of MM. We observed that the predominant subtype of MM among these individuals was IgA kappa, which diverges from the prevalent subtype, IgG kappa, observed in the general population. IgA is known to typically have more cytogenetic abnormalities and overall poorer prognosis, likely contributing to the difference in OS compared to patients with t-MM and MM not associated with prior malignancy. These insights shed light on the unique characteristics and potential influences of treatment-related MM in patients with prior malignancies or MPN.

Examining Racial Disparities in Multiple Myeloma Research and Treatment

Multiple myeloma (MM) is a disease which occurs in all races, geographic locations, and ethnicities, however the burden of this cancer is especially prominent in the African American/Black population with prevalence estimated to be as high as two to three times that of the White population. As a result MM is the most common hematological malignancy in Black patients. Despite a higher disease burden, this population suffers disproportionate rates of mortality. From 2017-2022 Black men had a 6% higher incidence rate than White men, but a 19% higher mortality rate. This disparity in outcomes between ethnic groups is likely multifactorial. Some explanations include novel therapy underutilization, or prevalence of comorbid conditions. It may also be attributable to biological differences, such as an increase rate of immunoglobin A disease, and an increased rate of chromosome 14 translocations. There may as a result be a heterogenous response to novel MM therapy. Unfortunately, clinical trial participation of Black patients has been significantly underrepresented. Studies that sought to analyze the results of novel therapy trials in this population found a trend towards decreased efficacy, however, were unable to reach significance due to insufficient study participation. We conducted a pooled analysis of interventional phase III clinical trials obtained from ClinicalTrials.gov data from 01/01/2000 to 12/31/2022. Studies who did not include racial/ethnicity data were excluded. There were 34 trials included in total that met this criteria and included in this analysis. There were 16,405 total enrolled patients analyzed. 13,618 (83.01%) were White, 1,255 (9.21%) were Asian, 777 (4.74%) were Black/African American, and 454 (2.7%) identified as Hispanic, with the remaining identified as other. Study participation was analyzed over time. Percentage of patients enrolled by year showed a negative correlation in Black/African Americans over the analyzed period. Our analysis suggests greater efforts need to be made to ensure the enrollment of a more diverse and representative MM population. Although this disease is roughly twice as prevalent in Black populations, clinical trial study participation since 2003 involved 18-times more White than Black patients. Despite ongoing recognition of this problem, study participation in MM has continued to remain largely homogenous. Interventions that may improve this problem include fostering and rebuilding trust in the clinical trial system that was damaged from historical abuses, expanding clinical sites to more diverse areas, expanding/revising eligibility criteria, and employing changes to enrollment techniques that better represent the demographics of the population intended to treat. Ultimately, the clinical trials for an intervention should appropriately reflect the population who will use it, otherwise these novel treatments may have unexpected heterogeneity in efficacy and safety once employed clinically.

Extracellular Vesicles Secreted from Daratumumab Resistant Cells Promote Resistance and Proliferation of Daratumumab Sensitive Cells, Possibly through the Transfer of miRNA Cargo

Multiple Myeloma (MM) is the second most common hematologic malignancy and remains incurable. Daratumumab (dara) is a potent anti-CD38 monoclonal antibody used for MM treatment, but responses are heterogeneous, and resistance is inevitable. There is hence an urgent need for identification of biomarkers to predict responses and in-depth characterisation of mechanisms of resistance. We have previously demonstrated that the exosome biogenesis pathway plays a significant role in the survival of dara resistant (dara R) cell lines. Here, we highlight that the enriched expression of exosome pathway proteins reported in dara R cell lines is not detected in Lenalidomide or Bortezomib-resistant cell lines, or in other monoclonal antibody resistant cell lines such as anti-CD20 (Rituximab)-resistant cell lines, thus indicating that this mechanism is unique to daratumumab resistance. Coculture of dara sensitive (dara S) cell lines with dara R cell lines separated by a cell-impermeable transwell insert promoted cell proliferation and reduced response to dara treatment in dara S cell lines thereby suggesting a potential role for extracellular vesicles (EVs) secreted by dara R cells in mediating these responses. In this study, we seek to investigate the role of EVs secreted by dara R cells, in particular, that of EV-derived miRNAs in the tumour microenvironment which can help predict and mediate dara resistance. We have originally described the establishment of three dara R cell lines in Natural Killer T-cell Lymphoma (NKTL), T-cell acute lymphoblastic leukaemia (T-ALL) and MM via long-term exposure to increasing concentrations of dara. To assess the specific role of EVs in mediating resistance, we isolated and purified EVs from the media of dara R cell lines and added it to the culture of dara S cells. To confirm uptake of dara R EVs by sensitive cell lines, EVs isolated from resistant cell lines were stained with PKH26 and characterised by binding to CD81 and CD63 beads via flow cytometry. Uptake of dara R EVs by dara S cell lines was confirmed through flow cytometry and confocal microscopy. We observed that uptake of dara R EVs directly promote proliferation and reduced cytotoxic response to dara treatment in dara S cells. Next, we assessed the effect of inhibiting dara R EV secretion by applying Neticonazole and Ketoconazole, prior to coculture of resistant and sensitive cell lines. Interestingly, coculturing azole-treated resistant cell lines with sensitive cell lines mitigated the increase in proliferation and dara resistance. Exosomal miRNAs are known to play a pivotal role in regulating cellular pathways driving cancer cell resistance and survival. To evaluate the potential role of EV-derived miRNA cargo, we first extracted miRNAs from the EVs purified from dara R and dara S cell lines and then performed an miRNA profiling screen with the PanoramiR panel (MiRXES) which targets 376 miRNAs expertly curated from miRBase 22, HMDD 3.2 and The Cancer Genome Atlas. We discovered that EVs derived from resistant cell lines expressed higher levels of proliferative miRNAs, miR-155 and miR-181a. These findings were validated through qPCR, confirming the increased expression of these miRNAs in both resistant cell lines and EVs derived from resistant cell lines. We also observed a concomitant downregulation of mir-155 target genes such as BACH1, JARID2 and SMAD5 in the resistant cell lines. This positive observation led us to extend the study into the clinic. Extracellular vesicles were isolated from the plasma fraction of bone marrow samples obtained from 17 patients with MM prior to treatment with dara based combinations either in the upfront or relapsed setting. miRNA extraction was performed and samples evaluated by miRNA sequencing. We found that the EVs from patients with a progression free survival (PFS) of less than 2 years after dara based therapy (dara R patients) had an enrichment of mir-155 and mir-181a as compared to those with a PFS longer than 2 years. These data align with our findings in the cell lines and suggest that these miRNAs may be utilised as potential biomarkers for daratumumab resistance. We are currently elucidating the biological basis for dara resistance mediated by these miRNAs. A deeper understanding of the mechanisms involved may pave the way for clinical evaluation of EV- directed therapeutics to overcome dara resistance.

The Activation Status of RSK2, but Not PDPK1 and AKT, Associates with the Prognosis of Diffuse Large B Cell Lymphoma

B-cell lymphoma (BCL) is the most common hematologic malignancy with various genetically and molecularly distinct subtypes. Although the prognosis of most BCL subtypes has been dramatically improved by the advances of disease subtype-directed, molecularly targeted therapies, some patients remain incurable with currently available therapeutics. We previously identified that a serine/threonine kinase 3-phosphoinositide-dependent kinase-1 (PDPK1) and its major downstream substrate serine/threonine kinases, AKT and RSK2, play pivotal roles in the pathophysiology of multiple myeloma and mantle cell lymphoma (MCL) (Chinen Y, Cancer Res 2014; Shimura Y, Mol Cancer Ther 2012; Matsumura-Kimoto Y, Cancer Med 2020). In particular, we found the crucial roles of the constitutive activation of the N-terminal kinase domain (NTKD) of RSK2 in cell survival, resistance to apoptosis, and cell proliferation of myeloma cells and MCL cells. However, the activation status of PDPK1, AKT, and RSK2-NTKD and their impacts on prognosis have yet to be comprehensively investigated in various subtypes of BCLs. To answer this, we here investigated the activation status of PDPK1, AKT, and RSK2-NTKD and their prognostic impacts in two major subtypes of BCLs, diffuse large B cell lymphoma (DLBCL) and follicular lymphoma (FL). To evaluate the activation status of the three kinases in DLBCL and FL, we performed the immunohistochemical analyses to characterize the expression profiles of phosphorylated (p)-PDPK1, p-AKT, and p-RSK2-NTKD in tumor-biopsied samples from 277 pts with DLBCL and 121 patients (pts) with FL diagnosed between 2010 and 2022 at our institution, and the expression levels of three target kinases were classified into four categories; negative (0), weak (1), moderate (2), and strong (3). As a result, in DLBCL, p-PDPK1, p-AKT, and p-RSK2 were positive in 98%, 68%, and 100%, respectively. More precisely, p-PDPK1 was 0-1 in 160 (59%) and 2-3 in 111 (41%). p-AKT was 0-1 in 225 (82%), and 2-3 in 48 (18%). p-RSK2 was 1 in 177 (65%), and 2-3 in 96 (35%). OS was significantly shorter in patients with moderate/strong expression (2-3) of p-RSK2 than those with weak expression (1) of p-RSK2 (5-year OS rate 52% vs. 73%, P<0.01), and, importantly, p-RSK2 status was identified as the independent prognostic factor by the multivariate analysis evaluating p-RSK2 expression and elements included in the International Prognostic Index as variables (HR 1.6, P=0.03). In addition, the prognostic relevance of RSK2 activity was evident in non-GCB type DLBCL but not GCB type DLBCL. In FL, p-PDPK1, p-AKT, and p-RSK2 were positive in 76%, 53%, and 69%, respectively. More precisely, p-PDPK1 status was 0-1 in 94 (89%) and 2-3 in 11 (10%), p-AKT status was 0-1 in 81 (77%) and 2-3 in 24 (23%)), and p-RSK2 was 0-1 in 67 (64%), and 2-3 in 38 (36%). In contrast to the case with DLBCL, the OS of FL was not associated with the activation status of p-PDPK1, p-AKT, and p-RSK2. In conclusion, the present analysis suggests the functional involvement of p-RSK2 activity and the role of future therapeutic targeting, especially in DLBCL, especially in high-risk non-GCB subtype.

An Idiotype Vaccine Strategy Utilizing Targeted NGS and Cell Free Protein Synthesis System Ensures Identification and Production of Human Idiotype Antigens, and Shows Promising Results in Preclinical Models of B-Cell Lymphoma with a Novel TLR7/8 Agonist-Based Adjuvant and Anti-PD-L1 Antibody

Background: Non-Hodgkin lymphoma (NHL) is one of the most common hematological malignancies in the world. Despite advances in treatment options, such as chimeric antigen receptor T cells (CAR-T) or bispecific antibodies, many patients still recur after complete remissions (CR) or do not achieve CR. Since B cell malignancies arise from clonal expansion, the B cell receptor (BCR) expressed on the cell surface, idiotype, has been studied as a tumor-specific antigen. Several trials have evaluated idiotype vaccines in clinical setting, but failed to demonstrate efficacy. Limitations have arisen from the uncertainty of BCR sequence confirmation, long turnaround time in manufacturing idiotype antigen, and the absence of strong immune modulators. Here, we utilized targeted next generation sequencing (NGS) for BCR, cell-free protein synthesis (CFPS) system for idiotype antigen manufacturing, and a novel liposomal TLR7/8 agonist-based adjuvant to optimize idiotype vaccine. Methods: To generate the idiotype antigens as single-chain variable fragment (scFv) from A20 lymphoma cell line and biopsy samples from NHL patients, variable regions of BCR heavy and light chain were sequenced by targeted NGS. Total RNA was isolated from A20 cells and tumor tissues from patients, and reverse transcription-polymerase chain reaction (RT-PCR) was performed using constant region-specific primers, resulting in the 1 st strand cDNA. Subsequently, the 2 nd strand cDNA was synthesized using V-gene specific primers and sequenced using an Illumina MiSeq sequencer. We applied targeted NGS to patients' tumor tissues from Asan Medical Center and Seoul National University Hospital to understand whether this method works in human tissues as well. The identified sequence was cloned into a linear DNA template and expressed by CFPS system. We optimized the CFPS system and purification process of idiotype antigen scFv from A20 cell line and human tissues. The liposomal adjuvant ProLNG-001 (TLR7/8 agonist 140 μg) and ProLNG-002 (TLR7/8 agonist 70 μg + TLR4 agonist 5 ug) were synthesized by microfluidics method. Idiotype antigen A20-scFv 50 μg was given alone or mixed with ProLNG-001 or -002. To investigate the efficacy of the idiotype vaccine, BALB/c mice were subcutaneously injected with 5×10 5 of A20 cells. Mice were subcutaneously vaccinated 4 times at 1-week intervals starting from day 2 after tumor inoculation. Atezolizumab 10 mg/kg was administered intraperitoneally 3 times a week for 3 weeks in the idiotype vaccine + immune checkpoint inhibitor combination group (Figure 1a). Results: Targeted NGS has confirmed BCR sequences from A20 cell line and dominant malignant B cell clone from patients' tumor with a success rate of over 90% when sufficient volume (more than 500 mm 3) of biopsy sample was provided for RNA extraction. Also, we were able to express the idiotype scFv antigens determined from 18 NHL patients' tumor samples using CFPS system with a 100% success rate (Figure 1b). The untreated mice (Group 1) showed a tumor growth pattern similar to that of Group 2 (vaccination with antigen only). Vaccination with idiotype antigen mixed with liposomal adjuvants, ProLNG-001 (Group 3) and ProLNG-002 (Group 4), significantly inhibited tumor growth. The combination of anti-PD-L1 antibody and idiotype antigen mixed with ProLNG-002 (Group 5) generated even better overall anti-tumor responses with 14 out of 15 mice showing no residual tumor at day 34 after tumor inoculation. Conclusion: Taken together, the idiotype vaccine strategy using targeted NGS for BCR, CFPS system for idiotype antigen manufacturing, and TLR7/8-agonist-based liposomal adjuvants showed highly effective results in the preclinical syngeneic A20 tumor model. Furthermore, the usage of immune checkpoint inhibitors combined with idiotype vaccine in B cell lymphoma may exert synergistic effects. The CFPS system can be applied to swift, robust and personalized manufacturing of idiotype antigen for individuals with different idiotype sequence in a GMP setting, which was not possible in prior studies. These results support further development of the idiotypic vaccine as a treatment option for non-Hodgkin's B cell lymphomas.

Dual-Targeting CD33/CD123 NANOBODY ® T Cell Engager Targeting Leukemia Blasts and Stem/Progenitor Cells in Relapsed AML

Acute myeloid leukemia (AML) is the most common adult hematologic malignancy. Relapse is common and salvage treatment with cytotoxic chemotherapy is rarely curative. Immunotherapy targeting surface antigens expressed in leukemia cells has emerged as an effective approach for AML treatment for patients who are older/unfit for or relapsed from/refractory to current regimens (Isidori A, et al. Frontiers Oncol. 2021). CD123 and CD33, two clinically validated targets in AML, are jointly expressed on blasts and leukemic stem cells (LSC) in >95% of AML patients. However, their expression is heterogenous between subclones and between patients which may impact the efficacy of single-targeting agents in certain patient populations. We recently reported a novel dual-targeting CD33/CD123 NANOBODY® T-cell engager (CD33/CD123-TCE) and demonstrated the anti-leukemia activity of CD33/CD123-TCE in AML with good safety profile in vivo (Dullaers M, et. al, JITC. 2022). Here we showed that CD33/CD123-TCE effectively targets AML blasts and LSC in relapsed AML. The cell surface expression profiling of samples from 55 patients with relapsed/refractory AML showed that all samples were positive for CD123 and CD33. The frequency of subpopulations in all samples co-expressing CD33 and CD123 is the most prominent, followed by single expression of CD123, and CD33. CD33-CD123- subset represented a minority. To investigate the anti-leukemia effect of CD33/CD123 TCE, we treated 18 primary samples for 7 days and assessed apoptosis by flow cytometry. Treatment of CD33/CD123 TCE at 2.5nM (low dose) and 25nM (high dose) induced more than 50% specific apoptosis in majority of bulk AML, with no significant difference between low and high dose of CD33/123 TCE. Analysis of cytotoxicity towards CD34+ AML-LSC revealed that CD33/CD123 TCE at low and high doses resulted in similar specific apoptosis of CD34+ cells, half of samples reached higher than 50% of apoptosis. Colony-forming assay in 2 primary samples further confirmed that CD33/CD123 TCE effectively inhibited the clonogenicity of primary AML cells expressing CD123 and CD33. While the average activity of CD33/CD123 TCE was comparable to single-targeting CD33-TCE and CD123-TCE reagents, in a subset of patient samples (11% of all samples), the dual-targeting CD33/CD123-TCE induced higher apoptosis in bulk and CD34+ AML cells compared to single-targeting controls, supporting utility of dual targeting. Concomitantly, CD3+ T cell expansion was observed in all dual- and single treatment groups, with a trend toward stronger amplification by CD33/CD123-TCE. Next, we examined AML and T cell phenotypes using mass cytometry (CyTOF) to characterize the impact of the TCE treatments. UMAP plus FlowSOM analysis revealed heterogenous subsets of LSC within samples and across patients. LSC subsets defined by CD34+ are known to co-express various surface markers associated with LSC-drug resistance and disease relapse including CD135 (FLT3), CD117 (c-kit), CLL-1, PD-L1 and TIM-3. These subsets were found highly sensitive to CD33/CD123-TCE, with subset CD34+CD38+CD117+PD-L1+CD123+ and CD34+CD38- showing greater sensitivity to CD33/CD123-TCE. Despite diverse expression of leukemic markers, CD33/CD123-TCE depleted CD34+ AML-LSC in all 3 samples tested by CyTOF, whereas single-targeting CD123-TCE and CD33-TCE were only effective in 2/3 samples. Analysis of T cell subsets revealed that dual- or single- targeting TCE expanded CD4+ helper and CD8+ cytotoxic subsets in all samples compared to control. CD4+ and CD8+ cells co-expressing CD69, TIM-3 or PD-1 were expanded by TCE in all three samples. Expansion of central and effector memory T cells defined by CCR7+CD45RA- and CCR7-CD45RA- in CD4+ and CD8+ subsets were seen in all treatment groups in 2 samples, whereas the highest expansion of CCR7-CD45RA-CD4+ effector memory subsets by CD33/CD123-TCE was seen in 1 sample. Taken together, the CD33/CD123 dual-targeting NANOBODY® TCE exhibits potent anti-AML activity and offers a broad patient coverage. Immunophenotypic profiling confirmed the effectiveness of CD33/CD123-TCE in eliminating various blast- and LSC-subsets, activating CD4+ and CD8+ T cells or expanding central/effector memory CD4+ and CD8+ T cells. These dual-targeting immune engagers may constitute an effective treatment option for patients who are older/unfit for or relapsed from/refractory to current regimens.

Biomarker Driven Strategies for Targeted Elimination of Hypoxia Adapted Lymphoma Cells

Large body of evidence suggests that hypoxia drives aggressive molecular features irrespective of cancer type. Non-Hodgkin lymphomas (NHL) are the most common hematologic malignancies characterized by widespread disease and frequent involvement of diverse hypoxic microenvironments (e.g., bone marrow, malignant effusions, large lymphoma masses). Impact of long-term hypoxia on the biology of lymphoma cells and its potential role in mediating drug resistance and disease relapses due to adaptive changes shaped by the hypoxic microenvironment remain only partially understood. In this study we analyzed impact of short- and long-term hypoxia on a panel of six lymphoma cell lines including diffuse large B-cell lymphoma (UPF4D, UPF8D), Burkitt lymphoma (Ramos, UPF9T), and mantle cell lymphoma (HBL2, MINO). Only 2 out of 8 tested lymphoma cell lines (Ramos, and HBL2) survived longer than 4 weeks under deep hypoxia (1% O2). The remaining tested lymphoma cell lines died by hypoxia-induced apoptosis (which could be inhibited by co-cultivation with pan-caspase inhibitor Z-VAD-FMK). The hypoxia-adapted (HA) Ramos and HBL2 cells had severely decreased proliferation rate accompanied by complex changes of the transcriptome, proteome, and metabolome. Seahorse analysis revealed significant inhibition of both oxidative phosphorylation and glycolytic pathways ( Figure 1). Of note, the whole-proteome profiling confirmed significant downregulation of proteins regulating oxidative phosphorylation, but significant upregulation of proteins regulating glycolysis. Sensitivity of HA cells to 2-deoxyglucose, an inhibitor of glycolysis was markedly increased ( Figure 1). The data suggest that under long-term deep hypoxia, lymphoma cells try to compensate for the decrease in ATP production from the oxidative phosphorylation process by boosting structural machinery of glycolysis, on which they became vitally dependent for survival. Despite the increased glycolytic machinary, however, the level of glycolysis decreases due to severe lack of oxygen. In our opinion, this is a clear representation of the Warburg effect, a hallmark of cancer based on the metabolic reprogramming of the cancer cells under hypoxia. Sensitivity of HA cells to a panel of the tested cytotoxic drugs (cytarabin, cisplatin, bortezomib) and targeted agents (venetoclax, S63545, A1155463, TRAIL, polatuzumab vedotin, IM-156, copanlisib) was either unchanged or increased (compared to sensitivity of the original cell lines cultured under normoxia). In contrast to so far published data, long-term hypoxia was not associated with acquired drug resistance in case of any of the tested agent. Of note, HA cells became significantly more sensitive to A1155463, an inhibitor of anti-apoptotic protein BCL-XL ( Figure 1). Unchanged levels of BCL-XL protein in HA cells and the respective normoxic cell lines (detected by western blotting) suggest that more complex mechanisms plausibly underlie the observed markedly increased sensitivity to A1155463. The whole-transcriptome and proteome analyses of both HA cell lines (compared to the parental cell lines cultured under normoxic conditions) detected among other complex changes signficant upregulation of P4HA1 (both mRNA, and protein), a prolyl hydroxylase involved in the stabilization of hypoxia-induced factor (HIF) 1 alpha, and putative oncogene associated with tumor aggressiveness. Transgenic (over)expression of P4HA1 in hypoxia-sensitive MINO cells effectively inhibited the hypoxia-induced apoptosis. Western blot analysis implemented on a panel of primary lymphoma cells revealed markedly higher expression of P4HA1 protein in the lymphoma cells obtained from malignant effusions (putative hypoxic microenvironment) compared to lymphoma cells obtained from leukemized blood (putative normoxic microenvironment). Our data suggest that P4HA1 positively impacts survival of lymphoma cells under hypoxia. In translation, P4HA1, BCL-XL, and structural proteins of the glycolytic pathway may represent novel drugable targets for more effective elimination of hypoxia-adapted lymphoma cells. Financial Support: Ministry of Health of the Czech Republic AZV NU23-03-00172, Grant Agency of the Czech Republic GA23-05377S, and National Institute for Cancer Research (EXCELES) LX22NPO5102.

The Role of Macrophage Migration Inhibitory Factor (MIF) in Regulation of T-Cell Activity in Multiple Myeloma

Background: Multiple myeloma (MM) is the second most common hematological malignancy associated with the overproduction of light chain or monoclonal immunoglobulin. MM remains incurable, with high relapses and refractory disease rates after first-line treatment. Hence, novel therapeutic approaches are needed to improve immune responses to prevent relapses or drug resistance after initial therapies. Immune responses are shaped by several factors, including mutations, pro- and anti-inflammatory cytokines and chemokines, and activation of immune checkpoints in the tumor microenvironment (TME) (Gonzalez et al., 2018). Macrophage migration inhibitory factor (MIF) as a soluble pro-inflammatory cytokine has become one of the most puzzling regulators of innate and adaptive immune responses. In myeloma, MIF was found to be significantly higher in MM cells from relapsed patients than those responding to the treatment. MIF has been implicated in drug resistance and suggested as a biomarker for predicting MM patient responses to proteasome inhibitors (Wang et al., 2020). We previously reported that MIF is highly expressed in MM and regulates CD84 (SLAMF5) expression in MM TME. We showed that MIF secreted from MM cells regulates PD-L1 expression through CD84 on CD14 + monocytic cells in the TME. Inhibition of MIF significantly reduced CD84 and PD-L1 expressions on human CD14 + cells and on myeloid-derived suppressor cells of the mice bearing MM (Lewinsky and Gunes et al., 2021). However, whether MIF directly regulates T-cell activity in MM TME has yet to be explored. Methods and Results: The current study reports that MIF may play a pivotal role in T-cell exhaustion in MM. Ouranalyses in published MM transcriptomic databases show that MM patients with low MIF had significantly longer overall and post-progression survival, indicating the therapeutic potential of MIF in MM. We also noted upregulated MIF gene expression in relapsed or refractory MM (R/RMM) patients (n=2) compared to that in newly diagnosed MM patients (n=7) and healthy individuals (n=9) in our and publicly available MM single-cell RNA sequencing datasets. We detected a profound MIF expression in MM cells and myeloid cell fractions, including monocytes, macrophages, and dendritic cells from the patients' bone marrow (BM) specimens. We previously reported that human MM cell lines highly express MIF. In this study, we assessed the impact of MIF secreted by MM cells on the immune cells, specifically CD8 + T cells. Our results showed that inhibition of MIF reduced the elevated T-cell exhaustion markers, PD1 and CTLA-4, on CD8 + T cells in healthy donors (HDs) peripheral blood mononuclear cells (PBMCs) following the coculturing with MM1.S cells (n=3 *P < 0.05 ). We also found that MIF inhibition induced the T-cell activation marker, LAMP1, on CD8 + T cells in this coculturing setting (n=3, **P < 0.01). Furthermore, we noted MIF inhibition reduced PD1 expression on CD8 + T cells in HDs PBMCs (n=3, **P < 0.01), suggesting MIF may regulate the exhaustion profile of CD8 + T cells. MIF can conduct paracrine or autocrine signalings by binding to its receptors, including CD74 and CD44, and lead to intracellular signaling cascades. Hence, we assessed CD74 gene expression as the counterpart of MIF in R/RMM, MM, and healthy patients. We noted significantly upregulated CD74 expression in R/RMM patients compared to newly diagnosed MM patients and healthy individuals. We also noted higher CD74 expression in CD8 + T cells than in CD4 + T cells in R/RMM patients, suggesting that MIF/CD74 axis may be a potential target to enhance CD8 + T cell-mediated immunity in R/RMM. To further explore the impact of MIF inhibition on MM progression, we performed a pilot in vivo study by inoculating murine 5TGM1 cells into two groups of syngeneic C57BL/KaLwRijHsd mice. After two weeks, the mice were injected with either sterile PBS as a control or a MIF inhibitor. The results of this pilot study showed that MIF inhibition significantly decreased MM cells in BM and PB of myeloma-bearing mice and reduced T-cell exhaustion markers on CD8 + T cells compared to that in the control mice (n=3-4, *P < 0.05, **P < 0.01, ***P < 0.001). Conclusion: Overall, our preliminary data indicate that MIF may have a crucial role in T-cell activity by regulating T-cell exhaustion markers in MM. Further studies are needed to explore the therapeutic potential of MIF to reduce T-cell exhaustion to improve anti-tumor immunity in MM.

The Potent Dihydroorotate Dehydrogenase Inhibitor, Hosu-53, Exhibits Compelling Monotherapy Efficacy in Multiple Myeloma and Augments CD47 Targeted Therapy

Worldwide, multiple myeloma (MM) is the second most common hematological malignancy characterized by the expansion of aberrant mature plasma cells in the bone marrow. Despite the FDA approval of several drugs with different mechanisms of action such as immunomodulatory drugs (IMiDs) and proteosome inhibitors (PIs), MM remains a challenging incurable disease where most patients ultimately relapse and become refractory to available . The resistant nature of MM underscores the need for novel therapeutic strategies that can directly affect MM cells while enhancing the outcome with MM current standard of care (SOC). Among the most promising MM SOC agents is monoclonal antibodies (mAb) such as the CD38 antibody daratumumab, which significantly improved the management of newly diagnosed and relapsed/refractory MM. Therefore, we aimed to develop novel therapies that can enhance mAb therapy in MM. Dihydroorotate dehydrogenase (DHODH) is an enzyme which mediates the fourth and rate-limiting step in the de novo pyrimidine synthesis pathway converting dihydroorotate to orotate, the precursor of uridine. Pyrimidine starvation using DHODH inhibitors has been shown to have potent antiproliferative activity in several malignancies including acute myeloid leukemia (AML) and MM. Thus, we developed a novel potent DHODH inhibitor, HOSU-53 for the treatment of hematological malignancies. We previously discovered the ability of HOSU-53 to modulate surface CD38 expression and found that HOSU-53 had potent monotherapy activity in MM and provided impressive synergy in combination with daratumumab using the NCI-H929 MM cell line derived xenograft (CDX) model (2022 AACR annual meeting abstracts). Herein, we further expanded our studies to pre-clinically develop HOSU-53 as a new potential MM therapy and explore additional synergistic immunotherapy combinations that could maintain efficacy in the case of anti-CD38 therapy resistance. We evaluated the in vitro antiproliferative efficacy of HOSU-53 against a panel of MM cell lines and found nanomolar potency validating that HOSU-53 would have monotherapy efficacy. Indeed, we conducted in vivo studies using two additional MM CDX subcutaneous (s.c) models, OPM-2 and RPMI-8226, and found significant tumor delay and survival advantage. In the OPM-2 model, HOSU-53 has a median survival of 54-days compared to vehicle at 28-days, while the median survival for HOSU-53 was 60-days compared to vehicle at 26-days in the RPMI-8226 model. We further verified HOSU-53 efficacy using the disseminated MM1.S luciferase CDX model and found a significant prolonged survival in the HOSU-53 cohort (median survival53-days) compared to vehicle (median survival28-days) that was further enhanced with isatuximab combination resulting in superior survival benefit (median survival 69-days). Given the continuous challenge in maintaining a durable response with anti-CD38 therapies due to resistance, we sought to explore additional combination regimens to maximize HOSU-53 efficacy. Currently there is significant clinical interest in CD47 antibody therapy such as magrolimab for both solid tumors and hematological malignancies. Our previous work suggested a strong synergy between CD47 antibodies and HOSU-53 in AML with curative potential (2022 ASH annual meeting abstracts), and hypothesized that we would observe similar synergy with HOSU-53 in combination with anti-CD47 in MM. Indeed, we found complete tumor regression in all mice treated with HOSU-53 + B6.H12 anti-CD47 therapy in the NCI-H929 s.c CDX model and significant prolonged survival and reduced bioluminescence in the NCI-H929 luciferase disseminated CDX model. Together, these two studies validate a potential clinical benefit to explore HOSU-53 + anti-CD47 regimen in MM patients. Furthermore, we observed that calreticulin (pro-phagocytosis signal) was modulated post HOSU-53 in vitro treatment suggesting its role in the observed synergy between HOSU-53 and CD47 blockade therapy. In summary, we show compelling survival benefit for HOSU-53 as a monotherapy which is further enhanced when combined with anti-CD38 or anti-CD47 therapies. HOSU-53 is expected to enter phase 1 clinical trials in 2024 and our data is supportive for its expansion into MM.

Characterization of a New Metabolic Score Correlated with CD38 Cell-Surface Expression and Response to Daratumumab Treatment in Multiple Myeloma

Multiple myeloma (MM) is the second most common hematological malignancy characterized by the uncontrolled accumulation of tumor plasma cells within the bone marrow. During the last 10 years, the development of new therapeutics, including the immune based therapies, significantly improved the life quality and survival of patients. However, the high molecular and clinical heterogeneity of this disease often leads to the development of resistance and relapses. Thus, MM unfortunately remains without definitive treatment in the majority of cases. Although few studies described the mechanisms of resistance to therapies developed by myeloma cells, a better understanding and new strategies to overcome the drug resistance mechanisms linked to Daratumumab remains of major interest for patients care. Among the factors that could be involved in myeloma cell resistance to anti-CD38 immunotherapies, we focused on the mitochondrial metabolism in MM, already described as a significant factor influencing response to treatments in several cancers. First, we defined a metabolic score using RNAseq data from different MM models, calculated with the gene expression of 112 genes involved either in the electron transport chain (Oxphos) or glycolysis. Each gene expression value was standardized and the metabolic score was built using the following equation: score= Σ(Glycolysis gene standardized expression) - Σ(Oxphos gene standardized expression). In our unique panel of 40 Human myeloma cell lines (HMCLs), high metabolic score significantly correlated with the functional metabolic profiles assessed by a Seahorse XFe96 analyzer included high mitochondrial and glycolysis activities on 25 HMCLs (r=0.6, p<0.05). Moreover, we tested the potential prognostic value of this metabolic score using cohorts of newly diagnosed MM patients treated by high dose of chemotherapy and autologous hematopoietic stem cell transplantation. In the MMRF CoMMpass cohort (674 patients), 32% of the patients with high metabolic score, defined using maxstat algorithm, were associated with a poor outcome (score>0.07; p<0.0005). These results were validated in an independent cohort of MM's patient (85 patients, Montpellier cohort), with a significant poor outcome associated with a high score (p<0.0005). Furthermore, we calculated the metabolic score in a cohort of MM's patients at diagnosis, who were then treated with anti-CD38 MoAb Daratumumab after relapse (Montpellier cohort N=42). Interestingly, we observed a significant difference between the mean score of patients with progressive or stable disease (n=13, mean score of 0.3380) and the mean score of patients with very good partial response or complete response (n=22, mean score of -0.0703), p<0.005). To validate these observations, the potential prognostic value was tested in that cohort: patients with high metabolic score were associated with a poor outcome (score>0.08; p<0.0005) suggesting a potential role of metabolism in the response to Dara treatment. We next questioned the possible link between the CD38 expression level at the surface of MM cells and their metabolic profile. For that purpose, we determined the CD38 surface antigen binding capacity (SABC) on 23 HMCLs representatives of MM heterogeneity. We observed a significant anti-correlation between the CD38 expression and the basal oxygen consumption rates (OCR) (r=-0.5, p<0.05) quantified with seahorse. Currently, correlations between CD38 expression measured on primary samples from patients with MM (MFI values) and their respective metabolic score are ongoing. However, these data suggest a link between mitochondrial activity, CD38 cell-surface expression and response to anti-CD38 MoAb. Altogether, our data demonstrated metabolism deregulation is associated with a prognostic value in newly diagnosed MM patients. Furthermore, we also reported a link between MM cell metabolism, CD38 expression and response to anti-CD38 MoAb treatment.

Single-Cell Multi-Ome Analysis Reveals Novel Molecular Mechanisms Underlying Subclonal Response to Survivin Inhibition in Relapsed/Refractory Multiple Myeloma

Multiple myeloma is the second-most common hematological malignancy in the US that remains a challenging disease to cure despite recent advancements in treatment strategies. Drug resistance is a major cause of concern in myeloma chemotherapy. We have created an optimization-regularization-based computational prediction method called secDrug that identified several novel secondary drug (‘secDrug‘) combinations against drug-resistant myeloma (relapsed/refractory multiple myeloma or RRMM). Top among our predicted secDrugs includes the survivin inhibitor, YM155. Survivin is a member of the inhibitor of apoptosis protein (IAP) family that has dual roles in mitosis and apoptosis. YM155 inhibits survivin at the transcriptional level by inhibiting the survivin gene promoter. Several studies have demonstrated the efficacy of YM155 in solid tumors and heme malignancies, including myeloma. However, the precise mechanism of action is not yet known. MM cell lines express high levels of IAPs. We observed high in vitro cytotoxicity of YM155 as a single agent (IC 50 range 0.41 to 12.85nM) as well as in combination with the primary drugs like proteasome inhibitors and Immunomodulatory drugs (IMiDs) against >15 human myeloma cell lines representing innate/refractory and acquired/emerging (relapse) resistance. Next, we explored the molecular mechanisms underlying the efficacy of YM155 using pre-vs-post-treatment tumor whole-transcriptome analysis (RNAseq). The top differentially regulated genes include DDIT3 (CHOP), ATF3, TXNDC5 SEC24D, and DHX15 ( Figure 1A). Ingenuity Pathway Analysis (IPA) predicted that, in addition to regulating apoptosis, survivin inhibition in myeloma significantly upregulated unfolded protein response and downregulated vehicle trafficking and genes associated with immune cell function. Further, we performed single-cell transcriptomics (scRNAseq; 10X Genomics) analysis to explore treatment-induced changes to the subclonal architecture. Our scRNAseq results revealed a distinct shift in clusters representing myeloma single-cell subpopulations following YM155 treatment ( Figure 1B). For example, Cluster 1 (53.11%), characterized by high expression of cell survival markers Ki67 and MYC, was pre-dominant in untreated myeloma cells, while Cluster 4 (52.19%) was the primary subclone in post-treatment tumors. Cluster 3 was shared between the untreated and treated groups. Detailed distinctions between each single-cell cluster will be presented. Furthermore, our scRNAseq data alluded to the benefits of combining survivin-inhibitors and Hsp90-inhibitors based on the expression of drug target genes. Hsp90 inhibitors indirectly target survivin by disrupting the interaction between survivin and Hsp90, resulting in a destabilized survivin protein that ultimately results in survivin degradation. In fact, we have earlier shown the effectiveness of Hsp90 inhibitors (17-AAG, AICAR, and CCT018159) as potent secDrugs against RRMM. Finally, high-dimensional mass cytometry or CyTOF analysis using heavy metal-conjugated proteins in patient bone marrow-derived primary myeloma cells ( ex vivo model system) showed that YM155 induced dose-dependent reduction of several protein markers implicated in cell survival and chemotherapeutic resistance in multiple myeloma, including myc, Ki-67, CD28, CD81, and CD274 (PD-L1). Previous studies have shown that CD28 and CD81 are high-risk immunophenotypes associated with significantly worse prognosis in patients with myeloma. PD-L1 (Programmed death-ligand 1) is a cell-surface glycoprotein expressed in a large number of malignancies that is involved in the termination of immune response and immune evasion. An earlier study has shown that YM155 enhances daratumumab-mediated cellular lysis of multiple myeloma cells. Our RNAseq and CyTOF results thus suggest a potential basis of synergy between YM155 with immunotherapies approved for myeloma, which we will test further. Our approach thus integrates in silico prediction with single-cell multi-ome analysis to identify molecular mechanisms potentially underlying subclonal response to novel combination therapy candidates (secDrugs) for the treatment of RRMM.

CAR-T Cell Therapy in the Treatment of Pediatric Non-Hodgkin Lymphoma.

Non-Hodgkin lymphomas (NHL) are a group of cancers that originate in the lymphatic system, especially from progenitor or mature B-cells, T-cells, or natural killer (NK) cells. NHL is the most common hematological malignancy worldwide and also the fourth most frequent type of cancer among pediatric patients. This cancer can occur in children of any age, but it is quite rare under the age of 5 years. In recent decades, available medicines and therapies have significantly improved the prognosis of patients with this cancer. However, some cases of NHL are treatment resistant. For this reason, immunotherapy, as a more targeted and personalized treatment strategy, is becoming increasingly important in the treatment of NHL in pediatric patients. The objective of the following review is to gather the latest available research results, conducted among pediatric and/or adult patients with NHL, regarding one immunotherapy method, i.e., chimeric antigen receptor (CAR) T cell therapy. We focus on assessing the effectiveness of CAR-T cell therapy, which mainly targets B cell markers, CD19, CD20, and CD22, their connections with one another, sequential treatment, or connections with co-stimulatory molecules. In addition, we also evaluate the safety, aftermath (especially neurotoxicities) and limitations of CAR-T cell therapy.

Current Status and Future of Artificial Intelligence in MM Imaging: A Systematic Review.

Artificial intelligence (AI) has attracted increasing attention as a tool for the detection and management of several medical conditions. Multiple myeloma (MM), a malignancy characterized by uncontrolled proliferation of plasma cells, is one of the most common hematologic malignancies, which relies on imaging for diagnosis and management. We aimed to review the current literature and trends in AI research of MM imaging. This study was performed according to the PRISMA guidelines. Three main concepts were used in the search algorithm, including "artificial intelligence" in "radiologic examinations" of patients with "multiple myeloma". The algorithm was used to search the PubMed, Embase, and Web of Science databases. Articles were screened based on the inclusion and exclusion criteria. In the end, we used the checklist for Artificial Intelligence in Medical Imaging (CLAIM) criteria to evaluate the manuscripts. We provided the percentage of studies that were compliant with each criterion as a measure of the quality of AI research on MM. The initial search yielded 977 results. After reviewing them, 14 final studies were selected. The studies used a wide array of imaging modalities. Radiomics analysis and segmentation tasks were the most popular studies (10/14 studies). The common purposes of radiomics studies included the differentiation of MM bone lesions from other lesions and the prediction of relapse. The goal of the segmentation studies was to develop algorithms for the automatic segmentation of important structures in MM. Dice score was the most common assessment tool in segmentation studies, which ranged from 0.80 to 0.97. These studies show that imaging is a valuable data source for medical AI models and plays an even greater role in the management of MM.